QP5"i +
H31
College of 1$l)v&itiau& anb ££>urgeotu(
|DR. WILLIAM J. GIES J?
| to enrich the library resources
available to holders ofthe
G1ES FELLOWSHIP
in Biological Chemistry
Digitized by the Internet Archive
in 2010 with funding from Columbia University Libraries
http://www.archive.org/details/practicalphysiolOOhawk
PRACTICAL PHYSIOLOGICAL CHEMISTRY
HAWK
Absorption Spectra.
PLATE I.
3 <&
Oxyhaemoglobln.
Haemoglobin.
Carboxy- haemoglobin.
Neutral Met-
haemoglobin.
Alkaline Met- haemoglobin.
Alkali Haematin.
Absorption Spectra.
PLATE H.
Reduced Alkali Haematin or Haemochromogen.
Acid Haematin in ethereal solution.
Acid Haemato- porphyrin.
Alkaline Haematopor-
phyrin.
Urobilin or Hydro- bilirubin in acid solution.
Urobilin or Hydro- bilirubin in alkaline solution after the addition of zinc chloride solution.
Bilicyanin or Cholecyanin in alkaline solution.
0 PRACTICAL
PHYSIOLOGICAL CHEMISTRY
A BOOK DESIGNED FOR USE IN COURSES IN PRACTICAL PHYSIOLOGICAL CHEMISTRY IN SCHOOLS OF MEDICINE AND OF SCIENCE
BY
PHILIP B. HAWK, M.S., Ph.D.
PROFESSOR OF PHYSIOLOGICAL CHEMISTRY IN THE UNIVERSITY OF ILLINOIS
WITH TWO FULL PAGE PLATES OF ABSORPTION SPECTRA IN COLORS, FOUR ADDITIONAL FULL PAGE COLOR PLATES AND ONE HUN- DRED AND TWENTY-SIX FIGURES OF WHICH TWELVE ARE FN COLORS
SECOND EDITION, REVISED AND ENLARGED
PHILADELPHIA
P. BLAKISTON'S SON & CO.
10I2 WALNUT STREET I9O9
PREFACE TO SECOND EDITION
The kind reception accorded this volume by the instructors in physiological chemistry in the United States and Great Britain has made the preparation of a new edition imperative, notwithstanding the fact that less than two years have elapsed since the former edition appeared. The advance and development made in the field of physiological chemistry during this period have been both rapid and important; conditions which would of themselves have neces- sitated the revision of the volume at an early date.
The book has been thoroughly revised in all departments and in part rewritten, the system of spelling officially adopted by the American Chemical Society having been followed throughout the volume. Besides introducing many neAv qualitative tests and quan- titative methods, the author has added a chapter on " Enzymes and Their Action " and has rewritten the two chapters on Proteins. The term " protein " has been substituted for " proteid " and the classification of proteins as recently adopted by the American Physi- ological Society and the American Society of Biological Chemists has been introduced and is followed throughout the text ; the classi- fication adopted by the British Medical Association is also included.
The original plan of the book has been adhered to with the excep- tion that the chapter on " Enzymes and Their Action " has been made Chapter I and the practical work upon the proteins is pre- ceded by a chapter giving a brief discussion of protein substances from the standpoint of their decomposition and synthesis. We believe that the student will be able to pursue his practical work more intelligently and will derive greater benefit therefrom if the plan of instruction as suggested in Chapters IV and V be followed in the presentation of the subject of " Proteins."
The author wishes to express his thanks to all those who so kindly offered suggestions for the betterment of the book. He is particu- larly desirous of expressing his gratitude to Professor Lafayette B. Mendel and Dr. Thomas B. Osborne for the many helpful sugges- tions they have so kindly given him. His thanks are also due Pro- fessor C. A. Herter, Dr. H. D. Dakin, Dr. S. R. Benedict and Mr. S. C. Clark for permission to insert unpublished material, to Mr. Paul E. Howe for valuable assistance rendered in the reading of
Vlll PREFACE TO SECOND EDITION.
proof and in the verification of tests and methods, and to Dr. M. E. Rehfuss for assistance in proof reading.
The author takes this opportunity of making an acknowledg- ment which was inadvertently omitted from the first edition. He wishes to express his obligation to the laboratories of physiological chemistry at Yale University and at Columbia University (College of Physicians and Surgeons) in the latter of which he was Assistant to Professor W. J. Gies for two years. The courses given in these laboratories formed the basis of many of the experiments included in this volume and it is with feelings of deepest gratitude that he records this acknowledgment of the assistance thus rendered by those in charge of these courses.
Philip B. Hawk.
Urbana, Illinois, Fehruary I, 1909.
PREFACE TO FIRST EDITION
The plan followed in the presentation of the subject of this volume is rather different, so far as the author is aware, from that set forth in any similar volume. This plan, however, he feels to be a logical one and has followed it with satisfactory results during a period of three years in his own classes at the University of Penn- sylvania. The main point in which the plan of the author differs from those previously proposed is in the treatment of the food stuffs and their digestion.
In Chapter IV the " Decomposition Products of Proteids " has been treated although it is impracticable to include the study of this topic in the ordinary course in practical physiological chemistry. For the specimens of the decomposition products, the crystalline forms of which are reproduced by original drawings or by micro- photographs, the author is indebted to Dr. Thomas B. Osborne, of New Haven, Conn.
Because of the increasing importance attached to the examina- tion of feces for purposes of diagnosis, the author has devoted a chapter to this subject. He feels that a careful study of this topic deserves to be included in the courses in practical physiological chemistry, of medical schools in particular. The subject of solid tissues (Chapters XIII, XIV and XV) has also been somewhat more fully treated than has generally been customary in books of this character.
The author is deeply indebted to Professor Lafayette B. Mendel, of Yale University, for his careful criticism of the manuscript and to Professor John Marshall, of the University of Pennsylvania, for his painstaking revision of the proof. He also wishes to express his gratitude to Dr. David L. Edsall for his criticism of the clinical portion of the volume ; to Dr. Otto Folin for suggestions regarding several of his quantitative methods, and to Mr. John T. Thomson for assistance in proof reading.
For the micro-photographs of oxyhemoglobin and haemin repro- duced in Chapter XI the author is indebted to Professor E. T. Reichert, of the University of Pennsylvania, who, in collaboration with Professor A. P. Brown, of the University of Pennsylvania, is making a very extended investigation into the crystalline forms of
X PREFACE TO FIRST EDITION.
biochemic substances. The micro-photograph of allantoin was kindly furnished by Professor Mendel. The author is also indebted for suggestions and assistance received from the lectures and pub- lished writings of numerous authors and investigators.
The original drawings of the volume were made by Mr. Louis Schmidt whose eminently satisfactory efforts are highly appreciated by the author.
Philip B. Hawk.
Philadelphia.
CONTENTS
CHAPTER I. Enzymes and Their Action i
CHAPTER II. Carbohydrates 21
CHAPTER III. Salivary Digestion 53
CHAPTER IV. Proteins: Their Decomposition and Synthesis 61
CHAPTER V. Proteins : Their Classification and Properties 85
CHAPTER VI. Gastric Digestion 118
CHAPTER VII. Fats 131
CHAPTER VIII. Pancreatic Digestion 140
CHAPTER IX. Bile 150
CHAPTER X. Putrefaction Products 162
CHAPTER XL Feces 172
CHAPTER XII. Blood 182
xi
Xll CONTENTS.
CHAPTER XIII. Milk 218
CHAPTER XIV. Epithelial and Connective Tissues 227
CHAPTER XV. Muscular Tissue 235
CHAPTER XVI. Nervous Tissue 248
CHAPTER XVII. Urine : General Characteristics of Normal and Path- ological Urine 254
CHAPTER XVIII. Urine : Physiological Constituents 264
CHAPTER XIX. Urine : Pathological Constituents 305
CHAPTER XX. Urine: Organized and Unorganized Sediments . . 343
CHAPTER XXI. Urine : Calculi 362
CHAPTER XXII. Urine : Quantitative Analysis 366
CHAPTER XXIII. Quantitative Analysis of Milk, Gastric Juice and
Blood 410
Index 427
LIST OF ILLUSTRATIONS
Plate
I. Absorption Spectral „ .. L.
TT . , . _r \- Frontispiece
11. Absorption Spectra J
III. Osazones Opposite page 24
IV. Normal Erythrocytes and Leucocytes Opposite page 184
V. Uric Acid Crystals Opposite page 273
VI. Ammonium Urate Opposite page 348
Figure Page
1. Dialyzing Apparatus for Students' Use 25
2. Einhorn Saccharometer 31
3. One Form of Laurent Polariscope 33
4. Diagrammatic Representation of the course of the Light
through the Laurent Polariscope 33
5. Polariscope (Schmidt and Hansen Model) 34
6. Iodoform 42
7. Potato Starch 45
8. Bean Starch 45
9. Arrowroot Starch 45
10. Rye Starch 45
11. Barley Starch 45
12. Oat Starch 45
13. Buckwheat Starch 45
14. Maize Starch 45
15. Rice Starch 45
16. Pea Starch 45
17. Wheat Starch 45
18. Microscopical Constituents of Saliva 56
19. Glycocoll Ester Hydrochloride 68
20. Serine 69
21. Phenylalanine 70
22. Fischer Apparatus 71
23. Tyrosine J2
24. Cystine y^
25. Histidine Dichloride 74
26. Leucine 76
xiii
XIV LIST OF ILLUSTRATIONS.
Figure Page
27. Lysine Picrate 78
28. Aspartic Acid 78
29. Glutamic Acid 79
30. Levo-a-Proline 80
31. Copper Salt of Proline . . . . . 81
32. Coagulation Temperature Apparatus 100
33. Edestin 104
34. Excelsin, the Protein of the Brazil Nut 105
35. Beef Fat 131
36. Mutton Fat '. 134
37. Pork Fat 136
38. Palmitic Acid 137
39. Melting-Point Apparatus 138
40. Bile Salts 152
41. Bilirubin (Hematoidin) 153
42. Cholesterol 159
43. Taurine 160
44. Glycocoll 161
45. Ammonium Chloride 166
46. Microscopical Constituents of Feces 172
47. Hematoidin Crystals from Acholic Stools 173
48. Charcot-Leyden Crystals 174
49. Boas' Sieve 177
50. Oxyhemoglobin Crystals from Blood of the Guinea Pig. 186
51. Oxyhemoglobin Crystals from Blood of the Rat 186
52. Oxyhemoglobin Crystals from Blood of the Horse 187
53. Oxyhemoglobin Crystals from Blood of the Squirrel . . . 187
54. Oxyhemoglobin Crystals from Blood of the Dog 188
55. Oxyhemoglobin Crystals from Blood of the Cat 188
56. Oxyhemoglobin Crystals from Blood of the Necturus . . 189
57. Effect of Water on Erythrocytes 195
58. Hemin Crystals from Human Blood 198
59. Hemin Crystals from Sheep Blood , 198
60. Sodium Chloride ' 200
61. Direct -vision Spectroscope 203
62. Angular-vision Spectroscope Arranged for Absorption
Analysis 204
63. Diagram of Angular-vision Spectroscope 204
64. Fleischl's TIemometer 208
65. Pipette of Fleischl's Hemometer 208
LIST OF ILLUSTRATIONS. XV
Figure Page
66. Colored Glass Wedge of Fleischl's Hiemometer 209
6y. Dare's Hsemoglobinometer 210
68. Horizontal Section of Dare's Hsemoglobinometer 211
69. Method of Filling the Capillary Observation Cell of
Dare's Hasmoglobinometer 212
70. Thoma-Zeiss Counting Chamber 213
71. Thoma-Zeiss Capillary .Pipettes 214
72. Ordinary Ruling of Thoma-Zeiss Counting Chamber ... 215
73. Zappert's Modified Ruling of Thoma-Zeiss Counting
Chamber 216
74. Normal Milk and Colostrum 219
75. Lactose 220
76. Calcium Phosphate 224
yy. Creatine 238
78. Xanthine 239
79. Hypoxanthine Silver Nitrate 245
80. Xanthine Silver Nitrate 247
81. Deposit in Ammoniacal Fermentation 257
82. Deposit in Acid Fermentation 257
83. Urinometer and Cylinder 258
84. Beckmann-Heidenhain Freezing-Point Apparatus 260
85. Urea 266
86. Urea Nitrate 268
87. Melting-Point Tubes Fastened to Bulb of Thermometer. 269
88. Urea Oxalate . . . 270
89. Pure Uric Acid 274
90. Creatinine 277
91. Creatinine-Zinc Chloride 278
92. Hippuric Acid 282
93. Allantoin from Cat's Urine 286
94. Benzoic Acid 289
95. Calcium Sulphate 298
96. " Triple Phosphate " .' 301
97. The Purdy Electric Centrifuge 343
98. Sediment Tube for the Purdy Electric Centrifuge 343
99. Calcium Oxalate 345
100. Calcium Carbonate 346
101. Various Forms of Uric Acid 347
102. Acid Sodium Urate 348
103. Cystine 349
XVI LIST OF ILLUSTRATIONS.
Figure Page
104. Crystals of Impure Leucine 350
105. Epithelium from Different Areas of the Urinary Tract. . 352
106. Pus Corpuscles 353
107. Hyaline Casts 354
108. Granular Casts 355
109. Granular Casts 356
1 10. Epithelial Casts ■ 356
in. Blood, Pus, Hyaline and Epithelial Casts 356
1 12. Fatty Casts 357
113. Fatty and Waxy Casts 357
1 14. Cylindroids ' 358
115. Crenated Erythrocytes 359
116. Human Spermatozoa 360
117. Esbach's Albuminometer 367
118. Marshall's Urea Apparatus 375
1 19. Hiifner's Urea Apparatus 377
120. Doremus-Hinds Ureometer 378
121. Folin's Urea Apparatus 379
122. Folin's Ammonia Apparatus 380
123. Folin Absorption Tube 381
124. Berthelot-Atwater Bomb Calorimeter 388
125. Soxhlet Apparatus 410
126. Feser's Lactoscope 41 1
PHYSIOLOGICAL CHEMISTRY.
CHAPTER I.
ENZYMES AND THEIR ACTION.
According to the old classification ferments were divided into two classes, the organized ferments and the unorganized ferments. As organized ferments or true ferments there were grouped such substances as yeast and certain bacteria which were supposed to act by virtue of vital processes, whereas the unorganized ferments included salivary amylase (ptyalin), gastric protease (pepsin), pancreatic protease (trypsin), etc., which were described as "non- living unorganized substances of a chemical nature." Kuhne des- ignated this latter class of substances as enzymes (iv typy — in yeast). This division into organized ferments (true ferments) and unorganized ferments (enzymes) was generally accepted and was practically unquestioned until Buchner overthrew it in the year 1897 by his epoch-making investigations on zymase. Previous to this time many writers had expressed the opinion that the action of the ferment organisms was similar to that of the unorganized ferments or enzymes and that therefore the activity of the former was possibly due to the production of a substance in the cell, which was in nature similar to an enzyme. Investigation after investiga- tion, however, failed to isolate any such principle from an active cell and the exponents of the " vital " theory became strengthened in their belief that certain fermentative processes brought about by living cells could not occur apart from the biological activity of such cells. However, as early as 1858, Traube had enunciated, in substance, the principles which were destined to be fundamental in our modern theory of fermentation. He expressed the belief that the yeast cell produced a product in its metabolic activities which had the property of reacting with sugar with the production of carbon dioxide and alcohol, and further that this reaction be- tween the product of the metabolism of the yeast cell and the sugar 2 1
2 PHYSIOLOGICAL CHEMISTRY.
occurred without aid from the original cell. It was not until 1897, however, that this theory was placed upon a firm experimental basis. This was brought about through the efforts of Buchner who succeeded in isolating from the living yeast cells a substance (zymase) which, when freed from the last trace of organized cel- lular material, was able to bring about the identical fermentative processes, which, up to this time, had been deemed possible only in the presence of the active, living yeast cell.
Buchner's manipulation of the yeast cells consisted in first grind- ing them with sand and infusorial earth after which the finely divided material was subjected to great pressure (300 atmospheres) and yielded a liquid which possessed the fermentative activity of the unchanged yeast cell.1 This liquid contained zymase, the prin- cipal enzyme of the yeast cell. Later the lactic-acid- and acetic- acid-producing bacteria were subjected by Buchner to treatment similar to that accorded the yeast cells, and the active intracellular enzymes were obtained. Many other instances are on record in which a soluble, active, agent has been isolated from ferment cells, with the result that it is pretty well established that all the so-called organized ferments elaborate substances of this character. Therefore, basing our definition on the work of Buchner and others we may define an enzyme, as an unorganised, soluble ferment, which is elaborated by an animal or vegetable cell and wihose ac- tivity is entirely independent of any of the life processes of such a cell. According to this definition the enzyme zymase elaborated by the yeast cell is entirely comparable to the enzyme pepsin elabor- ated by the cells of the stomach mucosa. One is derived from a vegetable cell, the other from an animal cell, yet the activity of neither is dependent upon the integrity of the cell.
Enzymes act by catalysis and hence may be termed catalyzers or catalysts. A simple rough definition of a catalyzer is " a sub- stance which alters the velocity of a chemical reaction without un- dergoing any apparent physical or chemical change itself and with- out becoming a part of the product formed." It is a well-known fact that the velocity of the greater number of chemical reactions may be changed through the presence of some catalyzer. For example, take the case of hydrogen peroxide. It spontaneously decomposes slowly into water and oxygen. In the presence of colloidal plati-
1 In later investigations the process was improved by freezing the ground cells with liquid air and finely pulverizing them, before applying the pressure.
ENZYMES AND THEIR ACTION. 3
num,1 however, the decomposition is much accelerated and ceases only when the destruction of the hydrogen peroxide is complete. Without multiplying instances, suffice it to say that there is an analogy between inorganic catalyzers and enzymes, the main point of difference between the enzymes and most of the inorganic cataly- zers being that the enzymes are colloids.2
Inasmuch as each of the enzymes has an action which is more or less specific in character, and since it is a fairly simple matter, ordinarily, to determine the character of that action, the classifi- cation of the enzymes is not attended with very great difficulties. They are ordinarily classified according to the nature of the sub- strate3 or according to the type of reaction they bring about. Thus we have various classes of enzymes such as amylolytic* proteolytic, lipolytic, glycolytic, mucolytic, autolytic, oxidizing, reducing, in- verting, protein-coagulating, deaniidizing, etc. In every instance the class name indicates the individual type of enzymatic activity which the enzymes included in that class are capable of accomplish- ing. For example, amylolytic enzymes facilitate the hydrolysis of starch (amylum) and related substances, lipolytic enzymes facilitate the hydrolysis of fats (Xi7ro?) whereas through the agency of uri- colytic enzymes, uric acid is broken down. There is a tendency, at the present time, to harmonize the nomenclature of the enzymes by the use of the termination, -ase. According to this system of nomenclature, all starch-transforming enzymes, or so-called amy- lolytic enzymes, are called amylases, all fat-splitting enzymes are called lipases, etc. Thus ptyalin the amylolytic enzyme of the saliva, would be termed salivary amylase in order to distinguish it from pancreatic amylase (amyl opsin) and vegetable amylases (di- astase, etc.). According to the same system, the fat-splitting enzyme of the gastric juice would be termed gastric lipase to dif- ferentiate it from pancreatic lipase (steapsin), the fat splitting enzyme of the pancreatic juice.
Our knowledge regarding the distribution of enzymes has been wonderfully broadened in recent years. Up to within a few years,
1 Produced by the passage of electric sparks between two platinum terminals immersed in distilled water, thus liberating ultra-microscopic particles.
2 Bredig has been able to obtain certain inorganic catalyzers in colloidal solution. These he calls "inorganic enzymes."
3 Substance acted upon.
4 Armstrong suggests the use of the termination " clastic " instead of " lytic." He calls attention to the fact that amylolytic} in analogy with electrolytic, means " decomposition by means of starch " and is therefore a misnomer. He sug- gests the use of aniyloclastic, protco clastic, etc.
4 PHYSIOLOGICAL CHEMISTRY.
the real scientific information as to the enzymes of the animal organism, for example, was limited, in the main, to a rather crude understanding of the enzymes intimately connected with the main digestive functions of the organism. We now have occasion to believe that enzymes are doubtless present in every animal cell and are actively associated with all vital phenomena. As a preeminent example of such cellular activity may be cited the liver cell with its reputed complement of 15-20 or more enzymes.
In text-book discussions of the enzymes it is customary to say that very little is known regarding the chemical characteristics of these substances since no member of the enzyme group has, up to the present time, been prepared in an absolutely pure condition. Apparently, however, from the nature of the facts in the case, it would be much more accurate to say that we absolutely do not knozv whether a specific enzyme lias, or has not, been prepared in a pure state. (Some authors, like Arthus, have assumed that enzymes are not chemical individuals, but properties conferred upon bodies.) The enzymes are very difficult to prepare in anything like a con- dition approximating purity, since they are very prone to change their nature during the process by which the investigator is attempt- ing to isolate them. For this reason we have absolutely no proof that the final product obtained is, or is not, in the same state of purity it possessed in the original cell. Some of the enzymes are more or less closely associated with the proteins from the fact that they are both formed in every cell as the result of the cellular acti- vity, both may be removed from solution by " salting-out," both are for the most part non-diffusible and are probably very similar as regards elementary composition. Hence in the preparation of some enzymes it is extremely difficult to make an absolute separation from the protein.1 Under certain conditions enzymes are readily adsorbed by shredded protein material, such as fibrin, and may successfully resist the most prolonged attempts at washing them free. We may summarize some of the properties of the great body of enzymes as follows : Enzymes are soluble in dilute glycerol, sodium chloride solution, dilute alcohol and water, and precipitable by ammonium sulphate and strong alcohol. Their presence may be proven from the nature of the end-products of their action and not through the agency of any chemical test. They are colloidal and non-diffusible ,and occur closely associated with protein material with which they possess many properties in common. Each enzyme
1 Others seem to be like the substrate on which they act, e. g., carbohydrate.
ENZYMES AND THEIR ACTION. 5
shows the greatest activity at a certain temperature called the optimum temperature; there is also a minimum and a maximum temperature for each specific enzyme. Their action is inhibited by sufficiently lowering the temperature, and the enzyme, if in solution, is entirely destroyed by subjecting it to a temperature of ioo° C. The best known enzymes, whether derived from warm-blooded or cold-blooded animals, are most active between 35°-45° C. The nature of the surrounding media alters the velocity of the enzymatic action, some enzymes being more active in acid solution whereas others require an alkaline fluid.
Many of the more important enzymes do not occur preformed within the cell, but are present in the form of a zymogen or mother- substance. In order to yield the active enzyme this zymogen must be transformed in a certain specific manner and by a certain specific substance. This transformation of the inactive zymogen into the active enzyme is termed activation. For instance, the zymogen of the enzyme pepsin of the gastric juice, termed pepsinogen, is acti- vated by the hydrochloric acid secreted by the gastric cells ( see p. 119), whereas the activation of the trypsinogen of the pancreatic juice is brought about by a substance termed entcrokinasc1 ( see p. 141). These are examples of many well known activation pro- cesses going on continually within the animal organism. The ag'ency which is instrumental in activating a zymogen is generally termed a zymo-excitcr or a kinase. In the cases cited hydrochloric acid would be termed a zymo-exciter and enterokinase would be termed a kinase.
After filtering yeast juice, prepared by the Buchner process (see p. 2), through a Martin gelatin filter. Harden and Young showed that the colloids left behind and the filtrate were both inactive fermentatively. Upon treating the colloid material (enzyme) with some of the filtrate, however, the mixture was shown to be able to bring about pronounced fermentation. It is believed that a co- enzyme present in the filtrate was the efficient agent in the trans- formation of the inactive enzyme. It is necessary to make frequent renewals of the co-enzyme in order to maintain continuous fermen- tation. It was further shown that this co-enzyme, in addition to being diffusible, was not destroyed by boiling and that it disappeared from yeast juice when this latter was fermented or allowed to undergo autolysis. The exact nature of this co-enzyme of zymase
1 According to Delezenne trypsinogen ma}- be rapidly activated by soluble calcium salts.
6 PHYSIOLOGICAL CHEMISTRY.
is unknown. The co-enzyme action, in this case, is probably de- pendent upon the presence of two individual agencies, one of which' is phosphates.
It has been shown by Loevenhart that the property of acting as a pancreatic lipase co-enzyme is vested in bile salts, and Magnus has further shown that the synthetic salts are as efficient in this regard as the natural ones. A few other instances of co-enzyme demonstrations have been reported.
The so-called " specificity " of enzyme action is an interesting and important fact. That enzymes are very specific as to the character of the substrate or substance acted upon, is well known. Emil Fischer investigated this problem of specificity extensively in connection with the fermentation of sugars and reached the conclusion that enzymes, with the possible exception of certain oxi- dases, can act only upon such substances as have a specific stereo- isomeric relationship to themselves. He considers that the enzyme and its substrate must have an interrelation, such as the key has to the lock, or the reaction does not occur. Fischer was able to predict, in certain definite cases, from a knowledge of the consti- tition and stereo-chemical relationships of a substance, whether or not it would be acted upon by a certain enzyme. An applica- tion of this specificity of enzyme action may be seen in the well- known facts that certain enzymes act on carbohydrates, others on fats, and others on protein, and moreover, that the group of those which transform carbohydrates, for example, is further subdivided into specific enzymes each of which has the power of acting alone upon some one sugar.'
It has been conclusively shown, in the case of certain enzymes,1 at least, that their action is a reversible one and is, in all its main features, directly analogous to the reversible reactions produced by chemical means. For instance, in the saponification of ethyl-buty- rate by means of pancreatic lipase, it has been shown that upon the formation of the end-products of the reaction, i. e., butyric acid and ethyl alcohol, there is reversion2 and the reaction is stationary. This does not mean that there are no chemical changes going on, but simply indicates that chemical equilibrium has been established,
1 This is probably a general condition.
2 The re-synthesis of ethyl-butyrate from its hydrolysis products. This may be indicated thus :
CsHtCOOGH, + H.O <=* C3HrCOOH + C2H5OH.
Ethyl butyrate. Butyric acid. Ethyl alcohol.
ENZYMES AND THEIR ACTION. /
and that the change in one direction is counterbalanced by the change in the opposite direction. Pancreatic lipase was one of the first enzymes to have the reversibility of its reaction clearly demon- strated.1 A knowledge of the fact that lipase possesses this rever- sibility of action is of extreme physiological importance and aids us materially in the explanation of the processes involved in the digestion, absorption and deposition of fats in die animal organ- ism (see p. 133).
In respect to many enzymes it has been found that the law gov- erning the action of inorganic catalyzers is directly applicable, i. e., that the intensity is almost directly proportional to the concentra- tion of the enzyme. In the case of enzymes, however, there is a difference in that a maximum intensity is soon reached and that subsequent concentration of the enzyme is productive of no further increase in intensity. The enzymes which have been shown to obey this linear lazv are lipase, invertase, rennin and trypsin. In certain instances, where this law of direct proportionality between the intensity of action and the concentration of enzyme does not hold, it has been found that the law of Schiitz, first experimentally demonstrated by E. Schiitz, was applicable. This is to the effect that the intensity is directly proportional to the square root of the concentration, or conversely, that the relative concentrations of enzymes are directly proportional to the squares of the intensities.2
It has been shown that there are certain substances which possess the property of directly inhibiting or preventing the action of a catalyzer. These are called anti-catalyzers or paralyzers and have been compared to the anti-toxins. Related to this class of anti- catalytic agents stand the anti-enzymes. The first anti-enzyme to be reported was the anti-rennin of Morgenroth. This was pro- duced by injecting into an animal increasing doses of rennet solu- tion, whereupon an " anti " substance was subsequently found both in the serum and in the milk, which prevented the enzyme rennin from exerting its normal activity in the presence of- caseinogen. In other words, anti-rennin had been formed in the serum of the animal,3 through the repeated injections of rennet solution. Since the discovery of this anti-enzyme, anti-bodies have been demon- strated for pepsin, trypsin, lipase, urease, amylase, laccase, tyro- sinase, emulsin, papain, and thrombin. According to Weinland,
1 The principle was first demonstrated in connection with the enzyme maltase (see p. 55).
2 This law of Schiitz is not generally applicable.
3 Serum is normally anti-try ptic.
5 PHYSIOLOGICAL CHEMISTRY.
the reason why the stomach does not digest itself is, that during life there is present in the mucous membrane of the stomach an anti-enzyme {anti-pepsin') which has the property of inhibiting the action of pepsin. A similar substance (anti-trypsin) is present in the intestinal mucosa as well as in the tissues of various intestinal worms. Some investigators are not inclined to accept the enzyme nature of these inhibitory agents as proven.
EXPERIMENTS ON ENZYMES AND ANTI-ENZYMES A. Experiments on Enzymes.1
I. AMYLASES.
i. Demonstration of Salivary Amylase.2 — To 25 c.c. of a one per cent starch paste in a small beaker, add 5 drops of saliva and stir thoroughly. At intervals of a minute remove a drop of the so- lution to one of the depressions of a test-tablet and test by the iodine test.3 If the blue color with iodine still forms after five minutes, add another five drops of saliva. The opalescence of the starch solution should soon disappear, indicating the formation of soluble starch (aniidulin) which gives a blue color with iodine. This body should soon be transformed into erythrodextrin which gives a red color with iodine and this, in turn, should pass into* achroodex- trin which gives no color with iodine. This point is called the achromic point. When this point is reached test by Fehling's test4 to show the production of a reducing substance (maltose). A posi- tive Fehling's test may be obtained while the solution still reacts red with iodine inasmuch as some sugar is formed from the soluble starch coincidently with the formation of the erythrodextrin. For further discussion of the transformation of starch see p. 54.
2. Demonstration of Pancreatic Amylase.5 — Proceed exactly as indicated above in the Demonstration of Salivary Amylase ex- cept that the saliva is replaced by 5 c.c. of pancreatic extract pre- pared as described on p. 144. Pancreatic amylase transforms the starch in a manner entirelv analogous to the transformation result- ing from the action of salivary amylase.
1If it is deemed advisable by the instructor to give all the practical work upon enzymes at this point in the course, additional experiments will be found in Chapters III, VI and VIII.
2 For a discussion of this enzyme see p. 54.
3 See p. 44. * See p. 27.
5 For a discussion of this enzyme see p. 142.
ENZYMES AND Till: Ik ACTION. 9
3. Preparation of Vegetable Amylase. — Extracl finely ground malt with water, filter and subject the filtrate to alcoholic fermenta- tion by means of yeast. When fermentation is complete filter off the yeast and precipitate the amylase from the filtrate by the addi- tion of alcohol. The precipitate may he filtered off and obtained in the form of a fine white powder.
4. Demonstration of Vegetable Amylase. — This enzyme may be demonstrated according to the directions given under Demonstra- tion of Salivary Amylase, p. 8, with the exception that the saliva used in that experiment is replaced by an aqueous solution of the vegetable amylase powder prepared as described above.1
II. PROTEASES.
i. Preparation of Gastric Protease.2 — Treat the finely com- minuted mucosa of a pig's stomach with 0.4 per cent hydrochloric acid and extract at 380 C. for 24-48 hours. The filtrate from this mixture constitutes a very satisfactory acid extract of gastric protease (see p. 122).
2. Demonstration of Gastric Protease. — Introduce some pro- tein material (fibrin, coagulated egg-white, or washed lean beef) into the acid extract of gastric protease prepared as above described.3 add an equal volume of 0.4 per cent hydrochloric acid and place the mixture at 38 ° C. for 2-3 days. Identify the products of digestion according to directions given on p. 122.
3. Preparation of Pancreatic Protease.4 — A satisfactory ex- tract of this enzyme may be made from the pancreas of a pig or sheep according to the directions given on p. 144.
4. Demonstration of Pancreatic Protease. — Into an alkaline extract of pancreatic protease,5 prepared as directed on p. 144. in- troduce some fibrin, coagulated egg-white or lean beef and place the mixture at 380 C. for 2-5 days.0 At the end of that period
1 If desired the first aqueous extract of the original malt may be used in this demonstration. Commercial taka-diastase may also be employed.
2 Also called pepsin, pepsase, gastric proteinase, and acid protease. For a dis- cussion of this enzyme see p. 120.
3 If so desired a solution of commercial pepsin powder in 0.2 per cent hydro- chloric acid may be substituted.
4 Also called trypsin, trypsasc. pancreatic proteinase and alkali proteinase. For a discussion of this enzyme see p. 141.
5 A 0.25 per cent sodium carbonate solution of commercial trypsin may bo substituted.
6 A few c.c. of toluene or an alcoholic solution of thymol should be added to prevent putrefaction.
IO PHYSIOLOGICAL CHEMISTRY.
separate and identify the end-products of the action of pancreatic protease according to the directions given on p. 145.
5. Demonstration of a Vegetable Protease. — A commercial preparation of papain (papayotin, carase or papase) , the protease of the fruit of the pawpaw (carica papaya), may be used in this connection. Follow the same procedure as that described under gastric protease (see p. 9).
III. LIPASES.
i. Preparation of Pancreatic Lipase.1 — An extract of this en- zyme may be prepared from the pancreas of the pig or sheep ac- cording to the directions given on p. 144.2
2. Demonstration of Pancreatic Lipase. — Into each of two test-tubes introduce 10 c.c. of milk and a small amount of litmus powder. To the contents of one tube add 3 c.c. of a neutral ex- tract of pancreatic lipase and to the contents of the other tube add 3 c.c. of a boiled neutral extract of pancreatic lipase. Keep the tubes at 38 ° C. and watch for color changes. The blue color of the litmus powder will gradually give place to a red. This change in the color of the litmus from blue to red has been brought about by the fatty acid which has been produced through the lipolytic ac- tion exercised by the lipase upon the milk fats.
3. Preparation of Vegetable Lipase. — This enzyme may be readily prepared from castor beans, several months old, by the fol- lowing procedure :3 Grind the shelled beans' very fine4 and ex- tract for twenty-four hour periods with alcohol-ether and ether, in turn. Reduce the semi-fat-free material to the finest possible con- sistency by means of mortar and pestle and pass this material through a sieve of very fine mesh. Place this material in a Soxhlet extractor and extract for one week. This fat-free powder may then be used to demonstrate the action of vegetable lipase. Powder prepared as described may be used in quantitative tests. For ordi- nary qualitative tests it is not necessary to remove the last traces of fat and therefore the extraction period in the Soxhlet apparatus may be much shortened.
1 Also called steapsin. For a discussion of this enzyme see p. 143. A very active lipolytic extract may also be prepared from the liver.
2 If preferred a glycerol extract may be prepared according to the directions given by Kanitz ; Zeitschrift fur physiologische Chemie, 1906, XLVI, p. 482.
3 A. E. Taylor: On Fermentation; University of California Publications, 1907. 1 The shells should be removed without the use of water. These beans are
poisonous due to their content of ricin.
ENZYMES AND THEIR ACTION. I I
4. Demonstration of Vegetable Lipase. — The lipolytic action of the lipase prepared from the castor bean, as just described, may be demonstrated in a manner entirely analogous to that used in the Demonstration of Pancreatic Lipase, see p. 10. Proceed as indi- cated in that experiment and substitute the vegetable lipase powder for the neutral extract of pancreatic lipase. The type of action is entirely analogous in the two instances.
An experiment similar to Experiment 2, p. 149, may also be tried if desired. In this experiment 0.2 c.c. of either ethyl batyrate or aniyl acetate may be employed.
IV. INVERTASES.1
i. Preparation of an Extract of Sucrase.2 — Treat the finely divided epithelium of the small intestine of a dog, pig, rat, rabbit, or hen, with about three volumes of a two per cent solution of sodium fluoride and permit the mixture to stand at room temperature for twenty-four hours. Strain the extract through cloth or absorbent cotton and use the strained material in the following demonstra- tion.
2. Demonstration of Sucrase. — To about 5 c.c. of a one per cent solution of sucrose, in a test-tube, add about one cubic centi- meter of a two per cent sodium fluoride intestinal extract, prepared as described above. Prepare a control tube in which the intestinal extract is boiled before being added to the sugar solution. Place the two tubes at 380 C. for two hours.3 Heat the mixture to boil- ing to coagulate the protein material, filter, and test the filtrate by Fehling's test (see p. 27). The tube containing the boiled extract should give no response to Fehling's test whereas the tube con- taining the unboiled extract should reduce the Fehling's solution. This reduction is due to the formation of invert sugar (see p. 41 ^ from the sucrose through the action of the enzyme sucrase which is present in the intestinal epithelium.
3. Preparation of Vegetable Sucrase. — Thoroughly grind about 100 grams of brewer's yeast in a mortar with sand. Spread the ground yeast in thin layers on glass or porous plates and dry it rapidly in a current of dry, warm air. Powder this dry yeast, extract it with distilled water and filter. Pour the filtrate into
1 The inverting enzymes of the alimentary tract; Mendel and Mitchell : American Journal of Physiology, 1907-08, XX, p. 81.
2 For a discussion of this enzyme see p. 144.
8 If a positive result is not obtained in this time permit the digestion to proceed for a longer period.
12 PHYSIOLOGICAL CHEMISTRY.
acetone, stir and after permitting the acetone mixture to stand for a few minutes filter on a Buchner funnel. The resulting precipi- tate, after drying and pulverizing, may be used to demonstrate vegetable sucrase.
4. Demonstration of Vegetable Sucrase. — To about 5 c.c. of a one per cent solution of sucrose in a test-tube add a small amount of the sucrase powder prepared as directed above. Place the tube at 380 C. for 24-72 hours and at the end of that period test the solution by Fehling's test. Reduction indicates that the active sucrase powder has transformed the non-reducing sucrose into dextrose and lsevulose, and these sugars, in turn, have reduced the Fehling solution.
5. Preparation of an Extract of Lactase.1 — Treat the finely divided epithelium of the small intestine of a kitten, puppy, or pig embryo with about three volumes of a two per cent solution of sodium fluoride and permit the mixture to stand at room tempera- ture for twenty- four hours. Strain the extract through cloth or absorbent cotton and use the strained material in the following demonstration.
6. Demonstration of Lactase.2 — To about 5 c.c. of a one per cent solution of lactose in a test-tube add about one cubic centi- meter of a toluene-water extract or a two per cent sodium fluoride extract of the first part of the small intestine3 of a kitten, puppy, or pig embryo prepared as described above. Prepare a control tube in which the intestinal extract is boiled before being added to the sugar solution. Place the two tubes at 38 ° C. for 24 hours. At the end of this period add one cubic centimeter of the digestion mixture to 5 c.c. of Barfoed's4 reagent and place the tubes in a boiling water-bath.5 Examine the tubes at the end of three min- utes against a black background in a good light. If no cuprous oxide is visible replace the tubes and repeat the examination at the end of the fourth and fifth minutes. If no reduction is then ob- served permit the tubes to stand at room temperature for 5-10 min- utes and examine again.6
1 For a discussion of this enzyme see p. 144. 3 Roaf ; Bio-Chemical Journal, 1908, III, p. 182.
3 Duodenum and first part of jejunum.
4 To 4.5 grams of neutral crystallized cupric acetate in 900 c.c. of water, add 0.6 c.c. of glacial acetic acid and make the total volume of the solution one liter.
5 Care should be taken to see that the water in the bath reaches at least to the upper level of the contents of the tubes.
6 Sometimes the drawing of conclusions is facilitated by pouring the mixture from the tube and examining the bottom of the tube for adherent cuprous oxide.
ENZYMES AND THEIR ACTION. 13
It has been determined that disaccharide solutions will not reduce' Barf oed's reagent until after they have been heated for 9-10 minutes on a boiling- water-bath in contact with the reagent.1 Therefore in the above test, if the tube containing the unboiled extract exhibits any reduction after being heated as indicated, for a period of five minutes or less, and the control tube containing boiled extract shows no reduction, it may be concluded that lactase was present in the intestinal extract.2
7. Preparation of an Extract of Maltase.3 — Treat the finely divided epithelium of the small intestine of a cat, kitten, or pig (embryo or adult) with about three volumes of a two per cent solu- tion of sodium fluoride and permit the mixture to stand at room temperature for twenty-four hours. Strain the extract through cloth and use the strained material in the following demonstration.
8. Demonstration of Maltase. — Proceed exactly as indicated in the demonstration of lactase, above, except that a one per cent solu- tion of maltose is substituted for the lactose solution. The extract used may be prepared from the upper part of the intestine of a cat, kitten, or pig (embryo or adult). In the case of lactase, as indi- cated, the intestine used should be that of a kitten, puppy, or pig (embryo).
V. EREPSIN.4
1. Preparation of Erepsin. — Grind the mucous membrane of the small intestine of a cat, dog or pig, with sand in a mortar. Treat the mortared membrane with toluene- or chloroform-water and permit the mixture to stand, with occasional shaking, for 24-72 hours.5 Filter the extract thus prepared through cotton and use the filtrate in the following experiment.
2. Demonstration of Erepsin. — To about 5 c.c. of a one per cent solution of Witte's peptone in a test-tube -add about one c.c. of the erepsin extract prepared as described above and make the mixture slightly alkaline (0.1 per cent) with sodium carbonate. Prepare a second tube containing a like amount of peptone solu- tion but boil the erepsin extract before introducing it. Place the
xThe heating for 9-10 minutes is sufficient to transform the disaccharide into monosaccharide.
2 The reduction would, of course, be due to the action of the dextrose and galactose which had been formed from the lactose through the action of the enzyme lactase.
3 For a discussion of this enzyme see p. 55.
4 Also called erepsase. For a discussion of this enzyme see p. 143.
5 The enzyme may also be extracted by means of glycerol or alkaline "physi- ological" salt solution if desired.
14 PHYSIOLOGICAL CHEMISTRY.
two tubes at 380 C. for 2-3 days. At the end of that period heat the contents of each tube to boiling, filter and try the biuret test on each filtrate. In making these tests care should be taken to use like amounts of filtrate, potassium hydroxide and cupric sulphate in each test in order that the drawing of correct conclusions may be facilitated. The contents of the tube which contained the boiled extract should show a deep pink color with the biuret test, due to the peptone still present. On the other hand the biuret test upon the contents of the tube containing the unboiled extract should be negative or exhibit, at the most, a faint pink or blue color, signify- ing that the peptone, through the influence of the erepsin, has been transformed, in great part at least, into amino acids which do not re- spond to the biuret test.1
VI. URICOLYTIC ENZYME.2
1. Preparation of Uricolytic Enzyme. — Extract pulped liver tissue with toluene- or chloroform-water at 380 C. for 24 hours, with occasional shaking. Filter the extract and use the filtrate in the following experiment.
2. Demonstration of Uricolytic Enzyme. — Add about 0.1 gram of uric acid to 10 c.c. of water and bring the uric acid into solution by the addition of the minimal quantity of potassium hy- droxide. To 5 c.c. of this uric acid solution, in a test-tube, add 5 c.c. of the uricolytic enzyme extract prepared as described above. Prepare a second tube containing a like amount of uric acid solution but boil the extract before it is introduced. Place the two tubes at 38 ° C. for 3-4 days and titrate the two digestive mixtures with a solution of potassium permanganate according to directions given under Folin-Shaffer Method, Chapter XXII. It will be found that the mixture containing the boiled extract requires a much larger volume of the permanganate to complete the titration than the other tube. This indicates that a uricolytic enzyme has destroyed at least a portion of the uric acid which was originally present in the tube containing the unboiled extract.
VII. CATALASE.
Demonstration of Catalase. — The various animal tissues, such as liver, kidney, blood, lung, muscle and brain contain an enzyme
1 Strictly speaking this erepsin demonstration is not adequate unless a control test is made with native protein (except caseinogen, histones and protamines) to show that the extract is trypsin-free and digests peptone but not native protein.
2 Mendel and Mitchell ; American Journal of Physiology, 1908, XX, p. 97.
ENZYMES AND THEIR ACTION. I 5
called caialase which possesses the property of decomposing hydro- gen peroxide. The presence of this enzyme may he demonstrated as follows: Introduce into a low, broad, wide-mouthed bottle some pulped liver tissue and a porcelain crucible containing neutral hydro- gen peroxide.1 Connect the bottle with a eudiometer filled with water, upset the crucible of hydrogen peroxide upon the liver pulp and note the collection of gas in the eudiometer. This gas is oxy- gen which has been liberated from the hydrogen peroxide through the action of the catalase of the liver tissue.
B. Experiments on Anti-Enzymes.
1. Preparation of an Extract of Anti-Pepsin.2-— Grind up a
number of intestinal worms (ascaris)3 with quartz sand in a mortar. Subject this mass to high pressure, filter the resultant juice and treat it with alcohol until a concentration of sixty per cent is reached. If any precipitate forms it should be filtered off4 and alcohol added to the filtrate until the concentration of alcohol is 85 per cent, or over. The anti-enzyme is precipitated by this concentration. Permit this precipitate to stand for twenty-four hours, then filter it off, wash it with 95 per cent alcohol, absolute alcohol, and ether, in turn, and finally dry the substance over sulphuric acid. The sticky powder which results may be used in this form or may be dissolved in water as desired and the aqueous solution used.5
2. Demonstration of Anti-Pepsin.6 — Introduce into a test-tube a few fibrin shreds and equal volumes of pepsin-hydrochloric acid7 and ascaris extract made as indicated above. Prepare a control tube in which the ascaris extract is replaced by water. Place the two tubes at 380 C. Ordinarily in one hour the fibrin in the control tube will be completely digested. The fibrin in the tube containing the ascaris extract may, however, remain unchanged for days, thus indicating the inhibitory influence exerted by the anti-enzyme pres- ent in this extract.
3. Preparation of an Extract of Anti-Trypsin.s — The extract
1 Mendel and Leavenworth; American' Journal of Physiology, 1908, XXI, p. 85. ~ Anti-gastric-protease or anti-acid-protease.
3 These may be readily obtained from pigs at a slaughter house.
4 This precipitate consists of impurities, the anti-enzyme not being precipitated until a higher concentration of alcohol is reached.
5 The original ascaris extract possesses much greater activity than either the powder or the aqueous solution.
6 Martin H. Fischer; Physiology of Alimentation, 1907, p. 134.
7 Made by bringing 0.015 gram of pepsin into solution in 7 c.c. of water and 0.23 gram of concentrated hydrochloric acid.
s Anti-pancreatic-protease or Anti-alkali-protease.
1 6 PHYSIOLOGICAL CHEMISTRY.
may be prepared from the intestinal worm, ascaris, according to the directions given on page 15.
4. Demonstration of Anti-Trypsin. — Introduce into a test-tube a few shreds of fibrin and equal volumes of an artificial tryptic solution1 and the ascaris extract made as described on page 15. Prepare a control tube in which the ascaris extract is replaced by water. Place the two tubes at 38 ° C. Ordinarily the fibrin in the control tube will be completely digested in two hours. The fibrin in the tube containing the ascaris extract may, however, remain unchanged for days, thus indicating the inhibitory influence of the anti-enzyme.
Blood serum also contains anti-try p sin. This may be demon- strated as follows : Introduce equal volumes of serum and artificial tryptic solution (prepared as described above) into a test-tube and add a few shreds of fibrin. Prepare a control tube containing boiled serum. Place the two tubes at 380 C. It will be observed that the fibrin in the tube containing the boiled serum digests, whereas that in the other tube does not digest. The anti-trypsin present in the unboiled serum has exerted an inhibitory influence upon the action of the trypsin.
C. Quantitative Applications.
1. Quantitative Determination of Amy loly tic Activity. — Wohl- gemuth^ Method. — Arrange a series of test-tubes with diminishing quantities of the enzyme solution under examination, introduce into each tube 5 c.c. of a 1 per cent solution of soluble starch2 and place each tube at once in a bath of ice water.3 When all the tubes have been prepared in this way and placed in the ice water-bath they are transferred to a water-bath or incubator and kept at 38 ° C. for from thirty minutes to an hour.4 At the end of this digestion
1 Made by dissolving 0.04 gram of sodium carbonate and 0.015 gram of trypsin in 8 c.c. of water.
2 Kahlbaum's soluble starch is satisfactory. In preparing the 1 per cent solu- tion, the weighed starch powder should be dissolved in the proper volume of cold, distilled water and stirred until a homogeneous suspension is obtained. The mixture should then be heated, with constant stirring, in a porcelain dish, until it is clear. This ordinarily takes about 8-10 minutes. A slightly opaque solution is thus obtained which should be cooled before using.
3 Ordinarily a series of six tubes is satisfactory, the volumes of the enzyme solution used ranging from 1 c.c. to 0.1 c.c. and the measurements being made by means of a 1 c.c. graduated pipette. Each tube should be placed in the ice water bath as soon as the starch solution is introduced. It will be found convenient to use a small wire basket to hold the tubes.
4 Longer digestion periods may be used where it is deemed advisable. If ex- ceedingly weak solutions are being investigated, it may be most satisfactory to permit the digestion to extend over a period of 24 hours.
ENZYMES AND THEIR ACTION. 17
period the tubes are again removed to the bath of ice water in order that the action of the enzyme may be stopped.
Dilute the contents of each tube, to within about one-half inch of the top, with water, add one drop of a T^ solution of iodine and shake the tube and contents thoroughly. A series of colors rang- ing from dark blue through bluish-violet and reddish-yellow to yellow, will be formed.1 The dark blue color shows the presence of unchanged starch, the bluish-violet indicates a mixture of starch and erythrodextrin, whereas the reddish-yellow signifies that eryth- rodextrin and maltose are present, and the yellow solution denotes the complete transformation of starch into maltose. Examine the tubes carefully before a white background and select the last tube in the series which shows the entire absence of all blue color, thus indicating that the starch has been completely transformed into dextrins and sugar. In case of indecision between two tubes, add an extra drop of the iodine solution, and observe them again, after shaking,
Calculation. — The amylolytic activity2 of a given solution is expressed in terms of the activity of i c.c. of such a solution. For example, if it is found that 0.02 c.c. of an amylolytic solution, acting at 380 C. completely transformed the starch in 5 c.c. of a 1 per cent starch solution in 30 minutes, the amylolytic activity of such a solution would be expressed as follows :
< = 25o.
This indicates that 1 c.c. of the solution under examination pos- sesses the power of completely digesting 250 c.c. of a 1 per cent starch solution in 30 minutes at 38 ° C.
2. Quantitative Determination of Peptic Activity, (a) Mett's Method. — The determination of the actual rate of peptic ac- tivity is a most important procedure under certain conditions. Sev- eral methods of making this determination are in use. The method of Sprigg3 is probably the most accurate method yet devised for this purpose. It is, however, too complicated and time-consuming for clinical purposes. The method of Mett, given below, is very simple although not strictly accurate. The procedure is as follows : To about 5 c.c. of the gastric juice under examination in a test-
1 See p. 44.
' Designated by " D " the first letter of " diastatic."
3 Sprigg: Zeitschrift fur physiologische Chemie, 1902. XXXV, p. 465.
1 8 PHYSIOLOGICAL CHEMISTRY.
tube add a section of a Mett tube1 and place the mixture at 380 C. for ten hours. At the end of this period, the tube should be re- moved from the gastric juice and the length of the column of coagulated albumin which has been digested, carefully determined by means of a low power microscope and a millimeter scale. In normal human gastric juice the upper limit is 4 mm. However, control tests should always be made to determine the digestibility of the coagulated albumin in artificial gastric juice inasmuch as this factor will vary with different albumin preparations.
In connection with this test Schiitz's law should be borne in mind. This principle is to the effect that the amount of proteolytic enzyme present in a digestion mixture is proportional to the square of the number of millimeters of albumin digested. Therefore a gastric juice which digests 2 mm. of albumin contains four times as much pepsin as a gastric juice which digests only 1 mm. of albu- min. And further, if the quantities of albumin digested are 2 mm. and 3 mm. respectively, the ratio between the pepsin values will be as 4 : 9.
It is claimed by Nirenstein and Schiff that the principle of Schutz does not apply to gastric juice unless this fluid be diluted with fifteen volumes of N/20 hydrochloric acid.
(b) Fuld and Levison's Method. — This test is founded upon the fact, shown by Osborne, that edestin when brought into solu- tion in dilute acid will change in its solubility, due to the contact with the acid, and that a protean called edestan, which is insoluble in neutral fluid, will be formed. The procedure is as follows : Dilute the gastric juice under examination with 20 volumes of water and introduce gradually decreasing volumes of the diluted juice into a series2 of narrow test-tubes about 1 cm. in diameter.
1 In the preparation of these tubes, egg-white is diluted with an equal volume of water, the precipitated globulin filtered off and the filtrate collected in a tall, narrow beaker or a large test-tube. A bundle of capillary tubes about 10 cm. in length and 2 mm. in diameter are now placed in this vessel in such a manner that they are completely submerged in the albumin solution. After an examination has shown that the tubes are completely filled with the albumin solution and that there are no interfering air-bubbles, the vessel and its con- tained tubes is heated for 5-15 minutes in a boiling water-bath, in order to coagulate the albumin. When this coagulation is complete, the tubes are re- moved, all albumin adhering to them is carefully cleaned off, and the tubes rendered air-tight by the application of sealing wax at either end. When needed for use, these tubes are cut into sections about 2 cm. in length.
2 The longer the series, the more accurate the deductions which may be drawn.
ENZYMES AND THEIR ACTION. 19
The measurements of gastric juice may conveniently be made with a one c.c. pipette which is accurately graduated in Moo c.c. Into the first tube in the series may be introduced one c.c. of gastric juice, and the tubes which follow in the series may receive vol- umes which differ, in each instance, from the volume introduced into the preceding tube by Moo, Yso, !•!<>• or Mo of a cubic centi- meter. Now rapidly introduce into each tube the same volume (e. g., 2 c.c.) of a i : iooo solution of edestin'1 and place the tubes at 40 ° C. for one-half hour. At the end of this time stratify ammonium hydroxide upon the contents of each tube,2 place the tubes in position before a black background and examine them carefully. The ammonium hydroxide, by diffusing into the acid fluid, forms a neutral zone and in this zone will be precipitated any undigested edestan which is present. Select the tube in the series which contains the least amount of gastric juice and which ex- hibits no ring, signifying that the edestan has been completely digested, and calculate the peptic activity of the gastric juice under examination on the basis of the volume of gastric juice used in this particular tube.
Calculation. — Multiply the number of c.c. of edestin solution used by the dilution to which the gastric juice was originally sub- jected and divide the volume of gastric juice necessary to com- pletely digest the edestan by this product. For example, if 2 c.c. of the edestin solution was completely digested by 0.25 c.c. of a 1 : 20 gastric juice we would have the following expression ; 0.25 -i- 20 X 2 or 1 : 160. This peptic activity may be expressed in several ways, e. g., (a) 1:160 pepsin; (b) 160 pepsin content; (c) 160 parts.
3. Quantitative Determination of Tryptic Activity. — Gross' Method. — This method is based upon the principle that faintly alka- line solutions of casein are precipitated upon the addition of dilute (1 per cent) acetic acid whereas its digestion products are not so precipitated. The method follows : Prepare a series of tubes each
1 This edestin should be prepared in the usual way (see p. 103), and brought into solution in a dilute hydrochloric acid of approximately the same strength as that which occurs normally in the human stomach. This may be conveniently made by adding 30 c.c. of fT) hydrochloric acid to 70 c.c. of water. Ordinarily it should not take longer than one minute to introduce the edestin solution into the entire series of tubes. However, if the edestin is added to the tubes in the same order as the ammonium hydroxide is afterward stratified, no appreciable error is introduced.
2 Making the stratification in the same order as the edestin solution was added.
20 PHYSIOLOGICAL CHEMISTRY.
containing 10 c.c. of a o. i per cent solution of pure, fat-free, casein,1 which has been heated to a temperature of 400 C. Add to the contents of the series of tubes increasing amounts of the trypsin solution under examination,2 and place them at 400 C. for fifteen minutes. At the end of this time remove the tubes and acidify the contents of each with a few drops of dilute (1 per cent) acetic acid. The tubes in which the casein is completely digested will remain clear when acidified while those tubes which contain undi- gested casein will become more or less turbid under these condi- tions. Select the first tube in the series which exhibits no turbidity upon acidification, thus indicating complete digestion of the casein, and calculate the tryptic activity of the enzyme solution under ex- amination.
Calculation. — The unit of tryptic activity is an expression of the power of 1 c.c. of the fluid under examination exerted for a period of fifteen minutes on 10 c.c. of a 0.1 per cent casein solution. For example, if 0.5 c.c. of a trypsin solution completely digests 10 c.c. of a 0.1 per cent solution of casein iii fifteen minutes the activity of that solution would be expressed as follows :
Tryptic activity = 1 -f- 0.5 = 2.
Such a trypsin solution would be said to possess an activity of 2. If 0.3 c.c. of the trypsin solution had been required the solution would be said to possess an activity of 3.3 i. e., 1 -^-0.3 = 3.3.
1 Made by dissolving one gram of Griibler's casein in a liter of 0.1 per cent sodium carbonate. A little chloroform may be added to prevent bacterial action.
2 The amount of solution used may vary from 0.1-1 c.c. The measurements may conveniently be made by means of a 1 c.c. graduated pipette.
CHAPTER II.
CARBOHYDRATES.
The name carbohydrates is given to a class of bodies which are an especially prominent constituent of plants and which are found also in the animal body either free or as an integral part of various proteins. They are called carbohydrates because they contain the elements C, H and O; the H and O being present in the proportion to form water. The term is not strictly appropriate inasmuch as there are bodies such as acetic acid, lactic acid and inosite which have H and O present in the proportion to form water, but which are not carbohydrates, and there are also true carbohydrates which do not have H and O present in this proportion, e. g., rhamnose, C6H1205.
Chemically considered, the carbohydrates are aldehyde or ketone derivatives of complex alcohols. Treated from this standpoint the aldehyde derivatives are spoken of as aldoses, and the ketone deriva- tives are spoken of as ketoses. The carbohydrates are also fre- quently named according to the number of oxygen atoms present in the molecule, e. g., trioses, pentoses and hexoses.
The more common carbohydrates may be classified as follows :
I. Monosaccharides.
i. Hexoses, CGH12O0.
(a) Dextrose.
(b) Lsevulose.
(c) Galactose. 2. Pentoses, C5H10O5.
(a) Arabinose.
(b) Xylose.
(c) Rhamnose (Methyl-pentose), C6H1205. II. Disaccharides, C12H22On.
i. Maltose.
2. Lactose.
3. Iso-Maltose.
4. Sucrose.
22 PHYSIOLOGICAL CHEMISTRY.
III. Trisaccharides, C1SH3201G.
i. Raffinose.
IV. Polysaccharides, (C6H10O5)x.
i. Starch Group.
(a) Starch.
(b) Inulin.
(c) Glycogen.
(d) Lichenin.
2. Gum and Vegetable Mucilage Group.
(a) Dextrin.
(b) Vegetable Gums.
3. Cellulose Group.
(a) Cellulose.
(b) Hemi-Cellulose.
Each member of the above carbohydrate classes, except the mem- bers of the pentose group, may be supposed to contain the group C6H10O5 called the saccharide group. The polysaccharides consist of this group alone taken a large number of times, whereas the disaccharides may be supposed to contain two such groups plus a molecule of water, and the monosaccharides to contain one such group plus a molecule of water. Thus, (C6H10O5)x = polysac- charide, (C6H10O5)2 -f- H20 = disaccharide, C6H10O5 -f- H20 = monosaccharide. In a general way the solubility of the carbo- hydrates varies with the number of saccharide groups present, the substances containing the largest number of these groups being the least soluble. This means simply that, as a class, the monosac- charides (hexoses) are the most soluble and the polysaccharides (starches and cellulose) are the least soluble.
MONOSACCHARIDES.
Hexoses, C6H1206.
The hexoses are monosaccharides containing six oxygen atoms in the molecule. They are the most important of the simple sugars, and two of the principal hexoses, dextrose and lsevulose, occur widely distributed in plants and fruits. Of these two hexoses, dextrose results from the hydrolysis of starch whereas both dextrose and lsevulose are formed in the hydrolysis of sucrose. Galactose, which with dextrose results from the hydrolysis of lactose, is also
CARBOHYDRATES. 23
an important hexose. These three hexoses are fermentable by yeast, and yield laevulinic acid upon heating with dilute mineral acids. They reduce metallic oxides in alkaline solution, are optically active, and extremely soluble. With phenylhydrazine they form characteristic osazones.
CH2OH
DEXTROSE, (CHOH)4.
CHO
Dextrose, also called glucose or grape sugar, is present in the blood in small amount and may also occur in traces in normal urine. After the ingestion of large amounts of sucrose, lactose or dex- trose, causing the assimilation limit to be exceeded, an alimentary glycosuria may arise. In diabetes mellitus very large amounts of dextrose are excreted in the urine. The following structural for- mula has been suggested by Victor Meyer for d-dextrose :
COH H - C - OH
HO - C - H
I H - C - OH
H - C - OH
CH2OH
(For further discussion of dextrose see section on Hexoses, page 22.)
Experiments on Dextrose.
1. Solubility. — Test the solubility of dextrose in the "ordinary solvents" and in alcohol. (In the solubility tests throughout the book we shall designate the following- solvents as the " ordinary solvents": H20 ; 10 per cent NaCl; 0.5 per cent Na2COs ; 0.2 per cent HC1; concentrated KOH; concentrated HC1.)
2. Molisch's Reaction. — Place approximately 5 c.c. of concen- trated H2S04 in a test-tube. Incline the tube and slowly pour down the inner side of it approximately 5 c.c. of the sugar solution to which 2 drops of Molisch's reagent (a 15 per cent alcoholic solution
24 PHYSIOLOGICAL CHEMISTRY.
of a-naphthol) has been added, so that the sugar solution will not mix with the acid. A reddish-violet zone is produced at the point of contact. The reaction is due to the formation of furfurol,
HC^CH
HC C-CHO,
\/ 0
by the acid. The test is given by all bodies containing a carbohy- drate group and is therefore not specific and, in consequence, of very little practical importance.
3. Phenylhydrazine Reaction. — Test according to one of the following methods: (a) To a small amount of phenylhydrazine mixture, furnished by the instructor,1 add 5 c.c. of the sugar solu- tion, shake well and heat on a boiling water-bath for one-half to three-quarters of an hour. Allow the tube to cool slowly and examine the crystals microscopically (Plate III, opposite). If the solution has become too concentrated in the boiling process it will be light-red in color and no crystals will separate until it is diluted with water.
Yellow crystalline bodies called osazones are formed from certain sugars under these conditions, in general each individual sugar giving rise to an osazone of a definite crystalline form which is typical for that sugar. It is important to remember in this connec- tion that of the simple sugars of interest in physiological chemistry, dextrose and kevulose yield the same osazone. Each osazone has a definite melting-point and as a further and more accurate means of identification it may be recrystal'lized and identified by the determi- nation of its melting-point and nitrogen content. The reaction tak- ing place in the formation of phenyldextr osazone is as follows :
C6H1206 + 2(H2N-NH-C6H5) =
Dextrose. Phenylhydrazine.
C6H10O4(NNHC6H5)2 + 2H20 + H2.
Phenyldextrosazone.
(b) Place 5 c.c. of the sugar solution in a test-tube, add 1 c.c. of
1 This mixture is prepared by combining one part of phenylhydrazine hydro- chloride and two parts of sodium acetate, by weight. These are thoroughly mixed in a mortar.
PLATE III.
OSAZONES.
Upper form, dextrosazone or lxvulosazone ; central form, maltosazone ; lower form.
lactosazone.
CARBOHYDRATES.
25
the phenylhydrazine-acetate solution furnished by the instructor,1 and heat on a boiling water-bath for one-half to three-quarters of an hour. Allow the liquid to cool slowly and examine the crystals microscopically (Plate III, opposite p. 24).
The phenylhydrazine test has been so modified by Cipollina as to be of use as a rapid clinical test. The directions for this test are given in the next experiment.
4. Cipollina's Test. — Thoroughly mix 4 c.c. of dextrose solu- tion, 5 drops of phenylhydrazine (the base) and y2 c.c. of glacial acetic acid in a test-tube. Heat the mixture for about one minute over a low flame, shaking the tube continually to prevent loss of fluid by bumping. Add 4-5 drops of sodium hydroxide (sp. gr. 1. 16), being certain that the fluid in the test-tube remains acid, heat the mixture again for a moment and then cool the contents of the tube. Ordinarily the crystals form at once, especially if the sugar solution possesses a low specific gravity. If they do not appear immediately allow the tube to stand at least 20 minutes before deciding upon the absence of sugar.
Examine the crystals under the microscope and compare them with those shown in Plate III, opposite page 24.
5. Precipitation by Alcohol. — To 10 c.c. of 95 per cent alcohol add about 2 c.c. of dextrose solution. Compare the result with that obtained under Dextrin, 7, page 49.
Fig. 1.
Dialyzixg Apparatus for Students' Use.
6. Iodine Test.— Make the regular iodine test as given under Starch, 5, page 44, and keep this result in mind for comparison with the results obtained later with starch and with dextrin.
7. Diffusibility of Dextrose. — Test the diffusibility of dextrose
1 This solution is prepared by mixing one part by volume, in each case, of glacial acetic acid, one part of water and two parts of phenylhydrazine (the base).
26 PHYSIOLOGICAL CHEMISTRY.
solution through animal membrane, or parchment paper, making a dialyzer like one of the models shown in Fig. I, p. 25.
8. Moore's Test. — To 2-3 c.c. of sugar solution in a test-tube add an equal volume of concentrated KOH or NaOH, and boil. The solution darkens and finally assumes a brown color. At this point the odor of caramel may be detected. This is an exceedingly crude test and is of little practical value. The brown color is due to the oxidation of the dextrose and the resulting formation of the potassium or sodium salts of certain organic acids which are formed as oxidation products.
9. Reduction Tests. — To their aldehyde or ketone structure many sugars owe the property of readily reducing alkaline solu- tions of the oxides of metals like copper, bismuth and mercury; they also possess the property of reducing ammoniacal silver solu- tions with the separation of metallic silver. Upon this property of reduction the most widely used tests for sugars are based. When whitish-blue cupric hydroxide in suspension in an alkaline liquid is heated it is converted into insoluble black cupric oxide, but if a reducing agent like certain sugars be present the cupric hydroxide is reduced to insoluble yellow cuprous hydroxide, which in turn, on further heating, may be converted into brownish-red or red cuprous oxide. These changes are indicated as follows :
OH
/ Cu s^Cu = 0 + H20.
^v Cupric oxide
^tt (black).
Cupric hydroxide (whitish-blue).
OH
/
Cu
\
OH
2Cu-OH + H20 = 0.
/
Cu
\
OH
f^TX Cuprous hydroxide
(yellow).
Cu-OH
L-
CARBOHYDRATES. 2J
Cu
X0 + H20.
OH
/
Cu
Cuprous hydroxide Cuprous oxide
(yellow). (brownish-red).
The chemical equations here discussed are exemplified in Trom- mer's and Fehling's tests.
(a) Trommer's Test. — To 5 c.c. of sugar solution in a test- tube add one-half its volume of KOH or NaOH. Mix thor- oughly and add, drop by drop, a very dilute solution of cupric sulphate. Continue the addition until there is a slight permanent precipitate of cupric hydroxide and in consequence the solution is slightly turbid. Heat, and the cupric hydroxide is reduced to yellow cuprous hydroxide or to brownish-red cuprous oxide. If the solution of cupric sulphate used is too strong a small brownish- red precipitate produced in a weak sugar solution may be entirely masked. On the other hand, particularly in testing for sugar in the urine, if too little cupric sulphate is used a light-colored pre- cipitate formed by uric acid and purine bases may obscure the brownish-red precipitate of cuprous oxide. The action of KOH or NaOH in the presence of an excess of sugar and insufficient copper will produce a brownish color. Phosphates of the alkaline earths may also be precipitated in the alkaline solution and be mistaken for cuprous hydroxide. Trommer's test is not very satis- factory.
(b) Fehling's Test. — To about 1 c.c. of Fehling's solution1 in a test-tube add about 4 c.c. of water, and boil. This is done to determine whether the solution will of itself cause the forma- tion of a precipitate of brownish-red cuprous oxide. If such a precipitate forms, the Fehling's solution must not be used. Add sugar solution to the warm Fehling's solution a few drops at a time and heat the mixture after each addition. The production of yellow cuprous hydroxide or brownish-red cuprous
1 Fehling's solution is composed of two definite solutions— a cupric sulphate solution and an alkaline tartrate solution, which may be prepared as follows :
Cupric sulphate solution = 34.65 grams of cupric sulphate dissolved in water and made up to 500 c.c.
Alkaline tartrate solution = 125 grams of potassium hydroxide and 173 grams ■of Rochelle salt dissolved in water and made up to 500 c.c.
These solutions should be preserved separately in rubber-stoppered bottles and mixed in equal volumes when needed for use. This is done to prevent deterioration.
28 PHYSIOLOGICAL CHEMISTRY.
oxide indicates that reduction has taken place. The yellow pre- cipitate is more likely to occur if the sugar solution is added rap- idly and in large amount, whereas with a less rapid addition of smaller amounts of sugar solution the brownish-red precipitate is generally formed.
This is a much more satisfactory test than Trommer's, but even this test is not entirely reliable when used to detect sugar in the urine. Such bodies as conjugate glycuronates, uric acid, nucieo- protein and homogentisic acid when present in sufficient amount may produce a result similar to that produced by sugar. Phos- phates of the alkaline earths may be- precipitated by the alkali of the Fehling's solution and in appearance may be mistaken for cuprous hydroxide. Cupric hydroxide may also be reduced to cuprous oxide and this in turn be dissolved by creatinine, a normal urinary constituent. This will give the urine under examination a greenish tinge and may obscure the sugar reaction even when a considerable amount of sugar is present.
(c) Benedict's Modifications of Fehling's Test. — First Modifi- cation.— To 2 c.c. of Benedict's solution1 in a test-tube add 6 c.c. of distilled water and 7-9 drops (not more) of the solution under examination. Boil the mixture vigorously for about 15-30 sec- onds and permit it to cool to room temperature spontaneously. (If desired this process may be repeated, although it is ordinarily un- necessary.) If sugar is present in the solution a precipitate will form which is often bluish-green or green at first, especially if the percentage of sugar is low, and which usually becomes yellowish upon standing. If the sugar present exceeds 0.06 per cent this precipitate generally forms at or below the boiling point, whereas if less than 0.06 per cent of sugar is present the precipitate forms more slowly and generally only after the solution has cooled.
Benedict claims that, whereas the original Fehling test will not serve to detect sugar when present in a concentration of less than 0.1 per cent that the above modification will serve to detect sugar when present in as small quantity as 0.015-0.02 per cent.
1 Benedict's modified Fehling solution consists of two definite solutions — a cupric sulphate solution and an alkaline tartrate solution, which may be pre- pared as follows :
Cupric sulphate solution = 34.65 grams of cupric sulphate dissolved in water and made up to 500 c.c.
Alkaline tartrate solution = 100 grams of anhydrous sodium carbonate and 173 grams of Rochelle salt dissolved in water and made up to 500 c.c.
These solutions should be preserved separately in rubber-stoppered bottles and mixed in equal volumes when needed for use. This is done to prevent deterioration.
CARBOHYDRATES. 29
The modified Fehling solution used in the above test differs from the original Fehling solution in that 100 grams of sodium car- bonate is substituted for the 125 grams of potassium hydroxide ordinarily used, thus forming a Fehling solution which is consid- erably less alkaline than the original. This alteration in the com- position of the Fehling solution is of advantage in the detection of sugar in the urine inasmuch as the strong alkalinity of the ordinary Fehling' solution has a tendency, when the reagent is boiled with a urine containing a small amount of dextrose, to decompose sufficient of the sugar to render the detection of the remaining portion exceedingly difficult by the usual technique. Benedict claims that for this reason the use of his modified solu- tion permits the detection of much smaller amounts of sugar than does the use of the ordinary Fehling solution. He has further modified his solution for use in the quantitative determination of sugar (see Chapter XXII).
Second Modification} — Very recently Benedict has further modi- fied his solution and has succeeded in obtaining one which does not deteriorate upon long standing.2 The following is the procedure for the detection of dextrose in solution : To five cubic centimeters of the reagent in a test-tube add eight (not more) drops of the solution under examination. Boil the mixture vigorously for from one to two minutes and then allow the fluid to cool spontaneously. In the presence of dextrose the entire body of the solution zvill be filled with a precipitate, which may be red, yellow or green in color, depending upon the amount of sugar present. If no dextrose is present, the solution will remain perfectly clear. (If urine is being tested, it may show a very faint turbidity, due to precipitated urates.) Even very small quantities of dextrose (o. 1 per cent)
1 Private communication from Dr. S. R. Benedict.
2 Benedict's new solution has the following composition :
Cupric sulphate 17.3 grams.
Sodium citrate 173.0 grams.
Sodium carbonate (anhydrous) 100.0 grams.
Distilled water to make 1 liter.
With the aid of heat dissolve the sodium citrate and carbonate in about 600 c.c. of water. Pour (through a folded filter paper if necessary) into a glass gradu- ate and make up to 850 c.c. Dissolve the cupric sulphate in about 100 c.c. of water and make up to 150 c.c. Pour the carbonate-citrate solution into a large beaker or casserole and add the cupric sulphate solution slowly, with constant stirring. The mixed solution is ready for use and does not deteriorate upon long standing:.
30 PHYSIOLOGICAL CHEMISTRY.
yield precipitates of surprising bulk with this reagent, and the posi- tive reaction for dextrose is the filling of the entire body of the solution with a precipitate, so that the solution becomes opaque. Since amount rather than color of the precipitate is made the basis of this test, it may be applied, even for the detection of small quan- tities of dextrose, as readily in artificial light as in daylight.
(d) Boettger's Test. — To 5 c.c. of sugar solution in a test-tube add 1 c.c. of KOH or NaOH and a very small amount of bismuth subnitrate, and boil. The solution will gradually darken and finally assume a black color due to reduced bismuth. If the test is made on urine containing albumin this must be removed, by boiling and filtering, before applying the test, since with albumin a similar change of color is produced (bismuth sulphide).
(e) Nylander's Test (Almen's Test). — To 5 c.c. of sugar solu- tion in a test-tube add one-tenth its volume of Nylander's reagent1 and heat for five minutes in a boiling water-bath.2 The solution will darken if reducing sugar is present and upon standing for a few moments a black color will appear. This color is due to the precipitation of bismuth. If the test is made on urine containing albumin this must be removed, by boiling and filtering, before applying the test, since with albumin a similar change of color is produced. Dextrose when present to the extent of 0.08 per cent, may be detected by this reaction. It is claimed by Bechold that Nylander's and Boettger's tests give a negative reaction with solutions containing sugar when mercuric chloride or chloroform is present. Other observers have failed to verify the inhibitory action of mercuric chloride and have shown that the inhibitory in- fluence of chloroform may be overcome by raising the tempera- ture of the urine to the boiling-point for a period of five minutes previous to making the test. Urines rich in indican, uroerythrin or hcemaio porphyrin, as well as urines excreted after the inges- tion of large amounts of certain medicinal substances, may give a darkening of Nylander's reagent similar to that of a true sugar reaction.
According to Rustin and Otto the addition of PtCl4 increases the delicacy of Nylander's reaction. They claim that this pro-
' Nylander's reagent is prepared by digesting 2 grams of bismuth subnitrate and a grams of Rochelle salt in 100 c.c. of a 10 per cent potassium hydroxide solution. The reagent is then cooled and filtered.
2 Hammarsten suggests that the mixture should be boiled 2-5 minutes (accord- ing to the sugar content) over a free flame and the tube then permitted to stand K minutes before drawing conclusions.
CARBOHYDRATES.
31
cedure causes the sugar to be converted quantitatively. No quan- titative method has yet been devised, however, based upon this principle.
A positive Nylander or Boettger test is probably due to the fol-
FlG. 2.
lowing reactions :
(a) Bi(OH)2NO, + KOH = Bi(OH)8 + KN03.
(b) 2Bi(OH)3 - 30 = Bi2 + 3H20.
10. Fermentation Test. — " Rub up " in a mortar about 20 c.c. of the sugar solution with a small piece of compressed yeast. Transfer the mixture to a saccharometer ( shown in Fig. 2) and stand it aside in a warm place for about twelve hours. If the sugar is fermentable, alcoholic fermen- tation will occur and carbon dioxide will collect as a gas in the upper portion of the tube. On the completion of fermentation introduce a little potassium hydroxide solu- tion into the graduated portion by means of a bent pipette, place the thumb tightly over the opening in the apparatus and in- vert the saccharometer. Explain the result.
11. Barfoed's Test. — Place .about 5 c.c. of Barfoed's solution1 in a test-tube and heat to boiling. Add dextrose solution slowly, a few drops at a time, heating after each addition. Reduction is indicated by the formation of a red precipitate. If the precipitate does not form upon continued boiling allow the tube to stand a few minutes and examine. Sodium chloride interferes with the reaction (Welker).
Barfoed's test is not a specific test for dextrose as is frequently stated, but simply serves to detect monosaccharides. Disac- charides will also respond to the test, according to Hinkel and Sherman, if the sugar solution is boiled sufficiently long, in contact with the reagent, to hydrolyze the disaccharide through the action of the acetic acid present in the Barfoed's solution.
12. Formation of Caramel. — Gently heat a small amount of
1 Barfoed's solution is prepared as follows : Dissolve 4.5 grams of neutral, crystallized cupric acetate in 100 c.c. of water and add 0.12 c.c. of 50 per cent acetic acid.
Einhorx Saccharometer.
32 PHYSIOLOGICAL CHEMISTRY.
pulverized dextrose in a test-tube. After the sugar has melted and turned brown, allow the tube to cool, add water and warm. The coloring' matter produced is known as caramel.
13. Demonstration of Optical Activity. — A demonstration of the use of the polariscope, by the instructor, each student being, required to take readings and compute the " specific rotation."
Use of the Polariscope.
For a detailed description of the different forms of polariscopes, the method of manipulation and the principles involved the student is referred to any standard text-book of physics. A brief descrip- tion follows : An ordinary ray of light vibrates in every direction. If such a ray is caused to pass through a " polarizing " Nicol prism it is resolved into two rays, one of which vibrates in every direction as before and a second ray which vibrates in one plane only. This latter ray is said to be polarized. Many organic sub- stances (sugars, proteins, etc.) have the power of twisting or rotat- ing this plane of polarized light, the extent to which the plane is rotated depending upon the number of molecules which the polar- ized light passes. Substances which possess this power are said to be " optically active." The specific rotation of a substance is the rotation expressed in degrees which is afforded by one gram of substance dissolved in 1 c.c. of water in a tube one decimeter in length. The specific rotation, (a)v, may be calculated by means of the following formula,
in which
d = sodium light.
a == observed rotation in degrees.
p = grams of substance dissolved in 1 c.c. of liquid. / = length of the tube in decimeters. If the specific rotation has been determined and it is desired to ascertain the per cent of the substance in solution, this may be obtained by the use of the following formula,
P =
(«V
The value of p multiplied by 100 will be the percentage of the substance in solution.
CARBOHYDKATKS.
33
An instrument by means of which the extent of the rotation may be determined is called a polariscope or polarimeter. Such an in- strument designed especially for the examination of sugar solutions is termed a saccharimeter or polarizing saccharimeter. The form of
Fig. 3.
One Form of Laurent Polariscope.
B, Microscope for reading the scale ; C, a vernier ; E, position of the analyzing Nicol prism ; H, polarizing Nicol prism in the tube below this point.
polariscope shown in Fig. 3, above, consists essentially of a long barrel provided with a Nicol prism at either end (Fig. 4, below). The solution under examination is contained in a tube which is placed between these two prisms. At the front end of the instru- ment is an adjusting eye-piece for focusing and a large recording
Fig. 4.
HO
^>
Diagrammatic Representation of the Course of the Light through the Laurent Polariscope. (The direction is reversed from that of Fig. 3, above.)
a, Bichromate plate to purify the light ; b, the polarizing Nicol prism ; c, a thin quartz plate covering one-half the field and essential in producing a second polarized plane ; d, tube to contain the liquid under examination ; e, the analyzing Nicol prism ; f and g, ocular lenses.
disc which registers in degrees and fractions of a degree. The light is admitted into the far end of the instrument and is polarizad by passing through a Nicol prism. This polarized ray then traverses the column of liquid within the tube mentioned above and if the sub-
4
34
PHYSIOLOGICAL CHEMISTRY.
stance is optically active the plane of the polarized ray is rotated to the right or left. Bodies rotating the ray to the right are called dextro-rotatory and those rotating it to the left Icevo-rotatory.
Within the apparatus is a disc which is so arranged as to be without lines and uniformly light at zero. Upon placing the opti-
Fig. 5.
Polariscope (Schmidt and Hansch Model).
cally active substance in position, however, the plane of polarized light is rotated or turned and it is necessary to rotate the disc through a certain number of degrees in order to secure the normal conditions, i. e., " without lines and uniformly light." The differ- ence between this reading and the zero is a or the observed rotation in degrees.
Polarizing saccharimeters are also constructed by which the per- centage of sugar in solution is determined by making an observa- tion and multiplying the value of each division on a horizontal slid- ing scale by the value of the division expressed in terms of dex- trose. This factor may vary according to the instrument.
CARBOHYDRATES. 35
CH2OH
LJEVULOSE, (CHOH)3.
CO CHoOH
As already stated, lsevulose, sometimes called fructose or fruit sugar, occurs widely disseminated throughout the plant kingdom in company with dextrose. Its reducing power is somewhat weaker than that of dextrose. Lsevulose does not ordinarily occur in the urine in diabetes mellitus, but has been found in exceptional cases. With phenylhydrazine it forms the same osazone as dextrose. With methylphenylhydrazine, lsevulose forms a characteristic methyl- phenyllsevulosazone.
(For a further discussion of lsevulose see the section on Hexoses,
p. 22.)
Experiments on L^vulose.
I— ii. Repeat these experiments as given under Dextrose, pages
23-3I-
12. Seliwanoff's Reaction. — To 5 c.c. of Seliwanoff's reagent1
in a test-tube add a few drops of a lsevulose solution and heat the mixture to boiling. A positive reaction is indicated by the produc- tion of a red color and the separation of a red precipitate. The latter may be dissolved in alcohol to which it will impart a striking red color.
If the boiling be prolonged a similar reaction may be obtained with solutions of dextrose or maltose.
13. Borchardt's Reaction. — To about 5 c.c. of a solution of lsevulose in a test-tube add an equal volume of 25 per cent hydro- chloric acid and a few crystals of resorcin. Heat to boiling and after the production of a red color, cool the tube under running water and transfer to an evaporating dish or beaker. Make the mixture slightly alkaline with solid potassium hydroxide, return it to a test tube, add 2-3 c.c. of acetic ether and shake the tube vig- orously. In the presence of lsevulose, the acetic ether is colored yellow. (For further discussion of the test see Chapter XIX.)
14. Formation of Methylphenyllaevulosazone. — To a solution
1 Seliwanoff's reagent may be prepared by dissolving 0.05 gram of resorcin in 100 c.c. of dilute (1:2) hydrochloric acid.
34
PHYSIOLOGICAL CHEMISTRY.
stance is optically active the plane of the polarized ray is rotated to the right or left. Bodies rotating the ray to the right are called dextro-rotatory and those rotating it to the left Icevo-rotatory.
Within the apparatus is a disc which is so arranged as to be without lines and uniformly light at zero. Upon placing the opti-
Fig. 5.
Polariscope (Schmidt and Hansch Model).
cally active substance in position, however, the plane of polarized light is rotated or turned and it is necessary to rotate the disc through a certain number of degrees in order to secure the normal conditions, i. e., " without lines and uniformly light." The differ- ence between this reading and the zero is a or the observed rotation in degrees.
Polarizing saccharimeters are also constructed by which the per- centage of sugar in solution is determined by making an observa- tion and multiplying the value of each division on a horizontal slid- ing scale by the value of the division expressed in terms of dex- trose. This factor may vary according to the instrument.
CARBOHYDRATES. 35
CH2OH
LiEVULOSE, (CHOH)8.
CO CHoOH
As already stated, laevulose, sometimes called fructose or fruit sugar, occurs widely disseminated throughout the plant kingdom in company with dextrose. Its reducing power is somewhat weaker than that of dextrose. Laevulose does not ordinarily occur in the urine in diabetes mellitus, but has been found in exceptional cases. With phenylhydrazine it forms the same osazone as dextrose. With methylphenylhydrazine, laevulose forms a characteristic methyl- phenyllaevulosazone.
(For a further discussion of laevulose see the section on Hexoses, p. 22.)
Experiments on L^vulose.
I— ii. Repeat these experiments as given under Dextrose, pages
23-31-
12. Seliwanoff's Reaction. — To 5 c.c. of Seliwanoff's reagent1
in a test-tube add a few drops of a laevulose solution and heat the mixture to boiling. A positive reaction is indicated by the produc- tion of a red color and the separation of a red precipitate. The latter may be dissolved in alcohol to which it will impart a striking red color.
If the boiling be prolonged a similar reaction may be obtained with solutions of dextrose or maltose.
13. Borchardt's Reaction. — To about 5 c.c. of a solution of laevulose in a test-tube add an equal volume of 25 per cent hydro- chloric acid and a few crystals of resorcin. Heat to boiling and after the production of a red color, cool the tube under running water and transfer to an evaporating dish or beaker. Make the mixture slightly alkaline with solid potassium hydroxide, return it to a test tube, add 2-3 c.c. of acetic ether and shake the tube vig- orously. In the presence of laevulose, the acetic ether is colored yellow. (For further discussion of the test see Chapter XIX.)
14. Formation of Methylphenyllaevulosazone. — To a solution
1 Seliwanoff's reagent may be prepared by dissolving 0.05 gram of resorcin in 100 c.c. of dilute (1:2) hydrochloric acid.
36 PHYSIOLOGICAL CHEMISTRY.
of 1.8 gram of lsevulose in 10 c.c. of water add 4 grams1 of methyl- phenylhydrazine and enough alcohol to clarify the solution. Intro- duce 4 c.c. of 50 per cent acetic acid and heat the mixture for 5-10 minutes on a boiling water-bath.2 On standing 15 minutes at room temperature, crystallization begins and is complete in two hours. By scratching the sides of the flask or by inoculation, the solution quickly congeals to form a thick paste of reddish yellow silky needles. These are the crystals of methylphenyllcevulosazone. They may be recrystallized from hot 95 per cent alcohol and melt at
153° C
CH2OH
GALACTOSE, (CHOH)4.
CHO
Galactose occurs with dextrose as one of the products of the hydrolysis of lactose. It is dextro-rotatory, forms an osazone with phenylhydrazine and ferments slowly with yeast. Upon oxida- tion with nitric acid galactose yields mucic acid, thus differentiat- ing this monosaccharide from dextrose and lsevulose. Lactose also yields mucic acid under these conditions. The mucic acid test may be used in urine examination to differentiate lactose and galactose from other reducing sugars.
Experiments on Galactose.
1. Tollens' Reaction. — To equal volumes of galactose solu- tion and hydrochloric acid (sp. gr. 1.09) add a little phloroglucin, and heat the mixture on a boiling water-bath. Galactose, pentose and glycuronic acid will be indicated by the appearance of a red color. Galactose may be differentiated from the two latter sub- stances in that its solutions exhibit no absorption bands upon spec- Iroscopical examinations.
2. Mucic Acid Test. — Treat 100 c.c. of the solution containing galactose with 20 c.c. of concentrated nitric acid (sp. gr. 1.4) and evaporate the mixture in a broad, shallow glass vessel on a boiling water-bath until the volume of the mixture has been reduced to about 20 c.c. At this point the fluid should be clear, and a .fine white precipitate of mucic acid should form. If the percentage of galactose present is low it may be necessary to cool the solution
1 3.66 grams if absolutely pure.
2 Longer heating is to be avoided.
CARBOHYDRATES. 37
and permit it to stand for some time before the precipitate will form. It is impossible to differentiate between galactose and lactose by this test, but the reaction serves to differentiate these two sugars from all other reducing sugars. Differentiate lactose from galac- tose by means of Barfoed's test (p. 31).
3. Phenylhydrazine Reaction. — Make the test according to directions given under Dextrose, 3 or 4, pages 24 and 25.
Pentoses, C5H10O5.
In plants and more particularly in certain gums, very complex carbohydrates, called pentosans, occur. These pentosans through hydrolysis by acids may be transformed into pentoses. Pentoses do not ordinarily occur in the animal organism, but have been found in the urine of morphine habitues and others, their occur- rence sometimes being a persistent condition without known cause. They are non- fermentable, have strong reducing power and form osazones with phenylhydrazine. Pentoses are an important constitu- ent of the dietary of herbivorous animals. Glycogen is said to be formed after the ingestion of these sugars containing five oxygen atoms. This, however, has not been conclusively proven. On distillation with strong hydrochloric acid pentoses and pentosans yield furfurol, which can be detected by its characteristic red reac- tion with aniline-acetate paper.
CH2OH
ARABINOSE, (CHOH)3.
CHO
Arabinose is one of the most important of the pentoses. The /-arabinose may be obtained from gum arabic, plum or cherry gum by boiling for several hours with 1-2 per cent sulphuric acid. This pentose is dextrorotatory, forms an osazone and has reducing power. The i-arabinose has been isolated from the urine and yields an osazone which melts at 166-1680 C.
Experiments on Arabinose.
1. Tollens' Reaction. — To equal volumes of arabinose solu- tion and hydrochloric acid (sp. gr. 1.09) add a little phloroglucin and heat the mixture on a boiling water-bath. Galactose, pentose or glycuronic acid will lie indicated by the appearance of a red
38 PHYSIOLOGICAL CHEMISTRY.
color. To differentiate between these bodies make a spectroscopic examination and look for the absorption band between D and E given by pentoses and glycuronic acid. Differentiate between the two latter bodies by the melting-points of their osazones.
Compare the reaction with that obtained with galactose (page 36) .
2. Orcin Test. — Repeat 1, using orcin instead of phloroglucin. A succession of colors from red through reddish-blue to green is produced. A green precipitate is formed which is soluble in amyl alcohol and has absorption bands between C and D.
3. Phenylhydrazine Reaction. — Make this test on the arabinose solution according to directions given under Dextrose, 3 or 4, pages 24 and 25.
CH2OH
xylose, (CHOH)3.
I CHO
Xylose, or wood sugar, is obtained by boiling wood gums with dilute acids as explained under Arabinose, page 37. It is dextro- rotatory and forms an osazone.
Experiments on Xylose. 1-3. Same as for arabinose (see page 37).
RHAMNOSE, C6H1205.
Rhamnose or methyl-pentose is an example of a true carbohydrate which does not have the H and O atoms present in the proportion to form water. Its formula is C0H12O5. It has been found that rhamnose when ingested by rabbits or hens has a positive influence upon the formation of glycogen in those organisms.
DISACCHARIDES, C12H22On.
The disaccharides as a class may be divided into two rather dis- tinct groups. The first group would include those disaccharides which are found in nature as such, e. g., sucrose and lactose and the second group would include those disaccharides formed in the hydrolysis of more complex carbohydrates, e. g., maltose, and iso-maltose.
CARBOHYDRATES. 39
The disaccharides have the general formula C]2H22On, to which, in the process of hydrolysis, a molecule of water is added causing the single disaccharide molecule to split into two monosaccharide (hexose) molecules. The products of the hydrolysis of the more common disaccharides are as follows :
Maltose — dextrose -J- dextrose. Lactose = dextrose -\- galactose. Sucrose = dextrose -\- lsevulose.
All of the more common disaccharides except sucrose have the power of reducing certain metallic oxides in alkaline solution, notably those of copper and bismuth. This reducing power is due to the presence of the aldehyde group ( — CHO) in the sugar molecule.
MALTOSE, CjaHaaOn.
Maltose or malt sugar is formed in the hydrolysis of starch through the action of an enzyme, vegetable amylase (diastase) , con- tained in sprouting barley or malt. Certain enzymes in the saliva and in the pancreatic juice may also cause a similar hydrolysis. Maltose is also an intermediate product of the action of dilute mineral acids upon starch. It is strongly dextro-rotatory, reduces metallic oxides in alkaline solution and is fermentable by yeast after being inverted (see Polysaccharides, page 43) by the enzyme maltase of the yeast. In common with the other disaccharides, maltose may be hydrolyzed with the formation of two molecules of monosac- charide. In this instance the products are two molecules of dex- trose. With phenylhydrazine maltose forms an osazone, maltosa- zone. The following formula represents the probable structure of maltose :
CH2OH CHO
CHOH CHOH
4q physiological chemistry.
Experiments on Maltose.
i-ii. Repeat these experiments as given under Dextrose, pages
23-3I-
ISO-MALTOSE, C^H^On.
Iso-maltose, an isomeric form of maltose, is formed, along with maltose, by the action of diastase upon starch paste, and also by the action of hydrochloric acid upon dextrose. It also occurs with maltose as one of the products of salivary digestion. It is dextro- rotatory and with phenylhydrazine gives an osazone which is char- acteristic. Iso-maltose is very soluble and reduces the oxides of bismuth and copper in alkaline solution. Pure iso-maltose is prob- ably only slightly fermentable.
LACTOSE, C^H^On.
Lactose or milk sugar occurs ordinarily only in milk, but has often been found in the urine of women during pregnancy and lactation. It may also occur in the urine of normal persons after the ingestion of unusually large amounts of lactose in the food. It has a strong reducing power, is dextro-rotatory and forms an osazone with phenylhydrazine. Upon hydrolysis lactose yields one molecule of dextrose and one molecule of galactose.
In the souring of milk the bacterium lactis and certain other micro-organisms bring about lactic acid fermentation by transform- ing the lactose of the milk into lactic acid,
H OH
H - C - C - COOH,
I I H H
and alcohol. This same reaction may occur in the alimentary canal as the result of the action of putrefactive bacteria. In the prepara- tion of kephyr and koumyss the lactose of the milk undergoes alco- holic fermentation, through the action of ferments other than yeast, and at the same time lactic acid is produced. Lactose and galactose yield mucic acid on oxidation with nitric acid. This fact is made use of in urine analysis to facilitate the differentiation of these sugars from other reducing sugars.
Lactose is not fermentable by pure yeast.
carbohydrates. 41
Experiments on Lactose. i— i i. Repeat these experiments as given under Dextrose, pages
23-31.
12. Mucic Acid Test. — Treat 100 c.c. of the solution containing lactose with 20 c.c. of concentrated nitric acid (sp. gr. 1.4) and evaporate the mixture in a broad, shallow, glass vessel on a boiling water-bath, until the volume of the mixture has been reduced to about 20 c.c. At this point the fluid should be clear, and a fine white precipitate of mucic acid should form. If the percentage of lactose present is low it may be necessary to cool the solution and permit it to stand for some time before the precipitate will appear. It is impossible to differentiate between lactose and galactose by this test, but the reaction serves to differentiate these two sugars from all other reducing sugars.
Differentiate lactose from galactose by means of Barfoed's test, page 31.
SUCROSE, CioHooOn.
Sucrose, also called saccharose or cane sugar, is one of the most important of the sugars and occurs very extensively distributed in plants, particularly in the sugar cane, sugar beet, sugar millet and in certain palms and maples.
Sucrose is dextro-rotatory and upon hydrolysis, as before men- tioned, the molecule of sucrose takes on a molecule of water and breaks down into two molecules of monosaccharide. The mono- saccharides formed in this instance are dextrose and lsevulose. This is the reaction :
G12R22Oxl + H20 = C6H1206 -f- C6H1206.
Sucrose. Dextrose. Lsevulose.
This process is called inversion and may be produced by bacteria, enzymes and certain weak acids. After this inversion the previously strongly dextro-rotatory solution becomes laevo-rotatory. This is due to the fact that the lsevulose molecule is more strongly lawo- rotatory than the dextrose molecule is dextro-rotatory. The product of this inversion is called invert sugar.
Sucrose does not reduce metallic oxides in alkaline solution and forms no osazone with phenylhydrazine. It is not fermentable directly by yeast, but must first be inverted by the enzyme sitcrase (invertase or inverting contained in the yeast. The probable struc- ture of sucrose may be represented by the following formula.
42
PHYSIOLOGICAL CHEMISTRY.
Note the absence of any" true sugar group or free ketone or alde- hyde group.
CH2OH
CHOH
CHO
-0 CH2OH
H
Sucrose.
Fig. 6.
Experiments on Sucrose.
i-ii. Repeat these experiments according to the directions given under Dextrose, pages 23-31.
12. Inversion of Sucrose. — To 25 c.c. of sucrose solution in a beaker add 5 drops of concentrated HC1 and boil one minute. Cool the solution, render alkaline with solid KOH and upon the resulting fluid repeat experiments 3 (or 4) and 9 as given under Dextrose, pages 24-26. Explain the results.
13. Production of Alcohol by Fermentation. — Prepare a strong (10-20 per cent) solution of sucrose, add a small amount
of egg albumin or commercial peptone and introduce the mixture into a bottle of appropriate size. Add yeast, and b)^ means of a bent tube inserted through a stopper into the neck of the bottle, con- duct the escaping gas into water. As fermentation proceeds readily in a warm place the escaping gas may be collected in a eudiometer tube and examined. When the activity of the yeast has prac- tically ceased, filter the contents of the bottle into a flask and distil the mixture. Catch the first portion of the distillate separately and test for alco- hol by one of the following reactions :
(a) Iodoform Test. — Render 2-3 c.c. of the distillate alkaline with potassium hydroxide solution and add a few drops of iodine
Iodoform. (Autenrietk.)
CARBOHYDRATES. 43
solution. Heat gently and note the formation of iodoform crystals. Examine these crystals under the microscope and compare them with those in Fig. 6, p. 42.
(b) Aldehyde Test.— Place 5 c.c. of the distillate in a test-tube, add a few drops of potassium dichromate solution, K2Cr207, and render it acid with dilute sulphuric acid. Boil the acid solution and note the odor of aldehyde.
TRISACCHARIDES, C1SH32016.
RAFFINOSE.
This trisaccharide, also called melitose or melitriose, occurs in cotton seed, Australian manna and in the molasses from the prepara- tion of beet sugar. It is dextro-rotatory, does not reduce Fehling's solution and is only partially fermentable by yeast.
Raffinose may be hydrolyzed by weak acids the same as the poly- saccharides are hydrolyzed, the products being lsevulose and meli- biose; further hydrolysis of the melibiose yields dextrose and galactose.
POLYSACCHARIDES, (C6H10O5)x.
In general the polysaccharides are amorphous bodies, a few, how- ever, are crystallizable. Through the action of certain enzymes or weak acids the polysaccharides may be hydrolyzed with the for- mation of monosaccharides. As a class the polysaccharides are quite insoluble and are non-fermentable until inverted. By inversion is meant the hydrolysis of disaccharide or polysaccharide sugars to form monosaccharides, as indicated in the following equations :
(a) C12H22On + H2O = 2(CfiH12O0).
(b) CflH10OB + H2O = C6H"12Oe.
STARCH, (C6H1005)x.
Starch is widely distributed throughout the vegetable kingdom, occurring in grains, fruits and tubers. It occurs in granular form, the microscopical appearance being typical for each individual starch. The granules, which differ in size according to the source, are composed of alternating concentric rings of granulose and cel- lulose. Ordinary starch is insoluble in cold water, but if boiled with water the cell walls are ruptured and starch paste results. In general starch gives a blue color with iodine.
42
PHYSIOLOGICAL CHEMISTRY.
Note the absence of any "true sugar group or free ketone or alde- hyde group.
CH2OH
CHOH
CHO
'/
-0 CH2OH
H
Sucrose.
Fig. 6.
Experiments on Sucrose.
i-ii. Repeat these experiments according to the directions given under Dextrose, pages 23-31.
12. Inversion of Sucrose. — To 25 c.c. of sucrose solution in a beaker add 5 drops of concentrated HC1 and boil one minute. Cool the solution, render alkaline with solid KOH and upon the resulting fluid repeat experiments 3 (or 4) and 9 as given under Dextrose, pages 24-26. Explain the results.
13. Production of Alcohol by Fermentation. — Prepare a strong (10-20 per cent) solution of sucrose, add a small amount
of egg albumin or commercial peptone and introduce the mixture into a bottle of appropriate size. Add yeast, and 03^ means of a bent tube inserted through a stopper into the neck of the bottle, con- duct the escaping gas into water. As fermentation proceeds readily in a warm place the escaping gas may be collected in a eudiometer tube and examined. When the activity of the yeast has prac- tically ceased, filter the contents of the bottle into a flask and distil the mixture. Catch the first portion of the distillate separately and test for alco- hol by one of the following reactions :
(a) Iodoform Test. — Render 2-3 c.c. of the distillate alkaline with potassium hydroxide solution and add a few drops of iodine
Iodoform. (Autenrieth.)
CARBOHYDRATES.
43
solution. Heat gently and note the formation of iodoform crystals. Examine these crystals under the microscope and compare them with those in Fig". 6, p. 42.
(b) Aldehyde Test.— Place 5 c.c. of the distillate in a test-tube, add a few drops of potassium dichromate solution, K2Cr207, and render it acid with dilute sulphuric acid. Boil the acid solution and note the odor of aldehyde.
TRISACCHARIDES, C1SH32010.
RAFFINOSE.
This trisaccharide, also called melitose or melitriose, occurs in cotton seed, Australian manna and in the molasses from the prepara- tion of beet sugar. It is dextro-rotatory, does not reduce Fehling's solution and is only partially fermentable by yeast.
Raffinose may be hydrolyzed by weak acids the same as the poly- saccharides are hydrolyzed, the products being lsevulose and meli- biose; further hydrolysis of the melibiose yields dextrose and galactose.
POLYSACCHARIDES, (C6H10O5)x.
In general the polysaccharides are amorphous bodies, a few, how- ever, are crystallizable. Through the action of certain enzymes or weak acids the polysaccharides may be hydrolyzed with the for- mation of monosaccharides. As a class the polysaccharides are quite insoluble and are non- fermentable until inverted. By inversion is meant the hydrolysis of disaccharide or polysaccharide sugars to form monosaccharides, as indicated in the following equations :
(a) (&)
CjoHooOj! -f- HoO
c6h10o5 + h;o
2(C6H1206)
C6H1206.
STARCH, (C6H10O5)x.
Starch is widely distributed throughout the vegetable kingdom, occurring in grains, fruits and tubers. It occurs in granular form, the microscopical appearance being typical for each individual starch. The granules, which differ in size according to the source, are composed of alternating concentric rings of granulose and cel- lulose. Ordinary starch is insoluble in cold water, but if boiled with water the cell walls are ruptured and starch paste results. In general starch gives a blue color with iodine.
44 PHYSIOLOGICAL CHEMISTRY.
Starch is acted upon by amylases, e. g., salivary amylase (ptyalin) and pancreatic amylase (amylopsin) , with the formation of soluble starch, erythro-dextrin, achroo-dextrins , maltose, iso-maltose and dextrose (see Salivary Digestion, page 54). Maltose is the principal end-product of this enzyme action. Upon boiling a starch solution with a dilute mineral acid a series of similar bodies is formed, but under these conditions dextrose is the principal end-product.
Experiments on Starch.
1. Preparation of Potato Starch. — Pare a raw potato, com- minute it upon a fine grater, mix with water, and "whip up" the pulped material vigorously before straining it through cheese cloth or gauze to remove the coarse particles. The starch rapidly settles to the bottom and can be washed by repeated decantation. Allow the compact mass of starch to drain thoroughly and spread it out on a watch glass to dry in the air. If so desired this preparation may be used in the experiments which follow.
2. Microscopical Examination. — Examine microscopically the granules of the various starches submitted and compare them with those shown in Figs. 7-17, page 45. The suspension of the granules in a drop of water will facilitate the microscopical examination.
3. Solubility. — Try the solubility of one form of starch in each of the ordinary solvents (see page 23). If uncertain regarding the solubility in any reagent, filter and test the filtrate with iodine solu- tion as given under 5 below. The production of a blue color would indicate that the starch had been dissolved by the solvent.
4. Iodine Test. — Place a few granules of starch in one of the depressions of a porcelain test-tablet and treat with a drop of a dilute solution of iodine in potassium iodide. The granules are colored blue due to the formation of so-called iodide of starch. The cellulose of the granule is not stained as may be seen by examining microscopically.
5. Iodine Test on Starch Paste.1 — Repeat the iodine test using the starch paste. Place 2-3 c.c. of starch paste2 in a test-tube, add
1 Preparation of Starch Paste. — Grind 2 grams of starch powder in a motar with a small amount of cold water. Bring 200 c.c. of water to the boiling-point and add the starch mixture from the mortar with continuous stirring. Bring again to the boiling-point and allow it to cool. This makes an approximate T per cent starch paste which is a very satisfactory strength for general use.
2 For this particular test a starch paste of very satisfactory strength may be made by mixing 1 c.c. of a 1 per cent starch paste with 100 c.c. of water.
Fig. 7
CARBOHYDRATES.
Fig. 8.
45
Pea. Wheat.
Starch Granules from Various Sources. (Leffman and Beam.)
46 PHYSIOLOGICAL CHEMISTRY.
a drop of the dilute iodine solution and observe the production of a blue color. Heat the tube and note the disappearance of the color. It reappears on cooling.
In similar tests note the influence of alcohol and of alkali upon the so-called iodide of starch.
The composition of the iodide of starch is not definitely known.
6. Fehling's Test. — On starch paste (see page 27).
7. Hydrolysis of Starch. — Place about 25 c.c. of starch paste in a small beaker, add 10 drops of concentrated HC1, and boil. By- means of a small pipette, at the end of each minute, remove a drop of the solution to the test-tablet and make the regular iodine test. As the testing proceeds the blue color should gradually fade and finally disappear. At this point, after cooling and neutralizing with solid KOH, Fehling's test (see page 27) should give a positive result due to the formation of a reducing sugar from the starch. Make the phenylhydrazine test upon some of the hydrolyzed starch. What sugar has been formed?
8. Influence of Tannic Acid. — Add an excess of tannic acid solution to a small amount of starch paste in a test-tube. The liquid will become strongly opaque and ordinarily a yellowish-white pre- cipitate is produced. Compare this result with the result of the similar experiment on dextrin (page 49).
9. Diffusibility of Starch Paste. — Test the diffusibility of starch paste through animal membrane or parchment paper, making a dialyzer like one of the models shown in Fig. 1, page 25.
INULIN, (C6H10O5)x.
Inulin is a polysaccharide which may be obtained as a white, odorless, tasteless powder from the tubers of the artichoke, elecam- pane or dahlia. It has also been prepared from the roots of chicory, dandelion and burdock. It is very slightly soluble in cold water and quite easily soluble in hot water. In cold alcohol of 60 per cent or over it is practically insoluble. Inulin gives a negative reac- tion with iodine solution. The " yellow " color reaction with iodine mentioned in many books is doubtless merely the normal color of the iodine solution. It is very difficult to prepare inulin which does not reduce Fehling's solution slightly. This reducing power may be due to an impurity. Practically all commercial preparations of inulin possess considerable reducing power.
Inulin is lsevo-rotatory and upon hydrolysis by acids or by the
CARBOHYDRATES. 47
enzyme inulase it yields the monosaccharide l^evulose which readily reduces Fehling's solution. The ordinary amylolytic enzymes occur- ring in the animal body do not digest inulin.
Experiments on Inulin.
1. Solubility. — Try the solubility of inulin powder in each of the ordinary solvents. If uncertain regarding the solubility in any reagent, filter and neutralize the nitrate if it is alkaline in reaction. Add a drop of concentrated hydrochloric acid to the filtrate and boil it for one minute. Render the solution neutral or slightly alkaline with solid potassium hydroxide and try Fehling's test. What is the significance of a positive Fehling's test in this connection ?
2. Iodine Test. — (a) Place 2-3 c.c. of the inulin solution in a test-tube and add a drop of dilute iodine solution. What do you observe ?
(b) Place a small amount of inulin powder in one of the depres- sions of a test-tablet and add a drop of dilute iodine solution. Is the effect any different from that observed above?
3. Molisch's Reaction. — Repeat this test according to directions given under Dextrose, 2, page 23.
4. Fehling's Test. — Make this test on the inulin solution accord- ing to the instructions given under Dextrose, page 27. Is there any reduction?1
5. Hydrolysis of Inulin. — Place 5 c.c. of inulin solution in a test-tube, add a drop of concentrated hydrochloric acid and boil it for one minute. Now cool the solution, neutralize it with con- centrated KOH and test the reducing action of 1 c.c. of the solution upon 1 c.c. of diluted (1:4) Fehling's solution. Explain the result.2
GLYCOGEN, (C6H10O5)x.
(For discussion and experiments see Muscular Tissue, Chapter XV.)
1 See the discussion of the properties of inulin, page 46.
2 If the inulin solution gave a positive Fehling test in the last experiment it will be necessary to check the hydrolysis experiment as follows : To 5 c.c. of the inulin solution in a test-tube add one drop of concentrated hydrochloric acid, neutralize with concentrated KOH solution and test the reducing action of i c.c. of the resulting solution upon I c.c. of diluted (1:4) Fehling's solu- tion. This will show the normal reducing power of the inulin solution. In case the inulin was hydrolyzed, the Fehling's test in the hydrolysis experiment should show a more pronounced reduction than that observed in the check experiment.
48 PHYSIOLOGICAL CHEMISTRY.
LICHENIN, (C6H10O5)x.
Lichenin may be obtained from Cetraria islandica (Iceland moss). It forms a difficultly soluble jelly in cold water and an opalescent solution in hot water. It is optically inactive and gives no color with iodine. Upon hydrolysis with dilute mineral acids lichenin yields dextrins and dextrose. It is said to be most nearly related chemically to starch. Saliva, pancreatic juice, malt diastase and gas- tric juice have no noticeable action on lichenin.
DEXTRIN, (C6H1005)x.
The dextrins are the bodies formed midway in the stages of the hydrolysis of starch by weak acids or an enzyme. They are amor- phous bodies which are easily soluble in water, acids and alkalis but are insoluble in alcohol or ether. Dextrins are dextro-rotatory and are not fermentable by yeast.
The dextrins may be hydrolyzed by dilute acids to form dextrose. With iodine one form of dextrin (erythro-dextrin) gives a red color. Their power to reduce Fehling's solution is questioned.
Experiments on Dextrin.
i. Solubility. — Test the solubility of pulverized dextrin in the ordinary solvents (see page 23).
2. Iodine Test. — Place a drop of dextrin solution in one of the depressions of the test-tablet and add a drop of a dilute solution of iodine in potassium iodide. A red color results due to the forma- tion of the red iodide of dextrin. If the reaction is not sufficiently pronounced make a stronger solution from pulverized dextrin and repeat the test. The solution should be slightly acid to secure the best results.
Make proper tests to show that the red iodide of dextrin is influenced by heat, alkali and alcohol in a similar manner to the blue iodide of starch (see page 46).
3. Fehling's Test. — See if the dextrin solution will reduce Feh- ling's solution.
4. Hydrolysis of Dextrin. — Take 25 c.c. of dextrin solution in a small beaker, add 5 drops of dilute hydrochloric acid, and boil. By means of a small pipette, at the end of each minute, remove a drop of the solution to one of the depressions of the test-tablet and
CARBOHYDRATES. 49
make the iodine test. The power of the solution to produce a color with iodine should rapidly disappear. When a negative reaction is obtained cool the solution and neutralize it with concentrated potas- sium hydroxide. Try Fehling's test (see page 27). This reaction is now strongly positive, due to the formation of a reducing sugar. Determine the nature of the sugar by means of the phenylhydrazine test (see pages 24 and 25).
5. Influence of Tannic Acid. — Add an excess of tannic acid solution to a small amount of dextrin solution in a test-tube. No precipitate forms. This result differs from the result of the similar experiment upon starch (see Starch, 8, page 46).
6. Diffusibility of Dextrin. — (See Starch, 9, page 46.)
7. Precipitation by Alcohol. — To about 50 c.c. of 95 per cent alcohol in a small beaker add about 10 c.c. of a concentrated dextrin solution. Dextrin is thrown out of solution as a gummy white precipitate. Compare the result with that obtained under Dextrose, 5, page 25.
CELLULOSE, (C6H10O5)x.
This complex polysaccharide forms a large portion of the cell wall of plants. It is extremely insoluble and its molecule is much more complex than the starch molecule. The best quality of filter paper and the ordinary absorbent cotton are good types of cellulose.
Experiments on Cellulose.
1. Solubility. — Test the solubility of cellulose in the ordinary solvents (see page 23).
2. Iodine Test. — Add a drop of dilute iodine solution to a few shreds of cotton on a test-tablet. Cellulose differs from starch and dextrin in giving no color with iodine.
3. Formation of Amyloid.1 — Add 10 c.c. of dilute and 5 c.c. of concentrated H2S04 to some absorbent cotton in a test-tube. When entirely dissolved (without heating) pour one-half of the solution into another test-tube, cool it and dilute with water. Amy- loid forms as a gummy precipitate and gives a brown or blue colora- tion with iodine.
After allowing the second portion of the acid solution of cotton to stand about 10 minutes, dilute it with water in a small beaker and boil for 15-30 minutes. Now cool, neutralize with solid KOH and
1 This body derives its name from amylum (starch) and is not to be con- founded with amyloid, the glycoprotein (page 106).
5
5o
PHYSIOLOGICAL CHEMISTRY.
test with Fehling's solution. Dextrose has been formed from the cellulose by the action of the acid.
4. Schweitzer's Solubility Test. — Place a little absorbent cotton in a test-tube, add Schweitzer's reagent,1 and stir the cellulose with' a glass rod. When completely dissolved- acidify the solution with acetic acid. An amorphous precipitate of cellulose is produced. Schweitzer's reagent is the only solvent for cellulose.
REVIEW OF CARBOHYDRATES.
In order to facilitate the student's review of the carbohydrates, the preparation of a chart similar to the appended model is recom-
MODEL CHART FOR REVIEW PURPOSES.
|
Carbohydrate. |
"o t/2 |
0> H 01 a -3 0 |
V H VI "11 u 0 0 s |
QJ H *u V 6 s 0 H |
01 H "bo a o> fa |
H V V 0 M |
01 H V T3 C rt |
V H -a o> ,o |
(A 0 0 rt 0 .2 o> |
J" a J3 0 0 .3 ■3 « C 0> |
0) H "G <! 3 |
Is |
e . .2-0 S-8 0" 01 a, |
O) a q O |
c s 0 |
1 5 |
c 0 rt C 0) 6 fa |
J2 a B |
|
Dextrose. Lsevulose. |
|
|
— |
~ |
|
|
— |
|
— |
— |
— |
|||||||
|
Maltose. |
|
|||||||||||||||||
|
Iso-maltose. |
||||||||||||||||||
|
Lactose. |
|
|
|
|||||||||||||||
|
Sucrose. |
||||||||||||||||||
|
Starch. |
|
|||||||||||||||||
|
Inulin. |
|
|||||||||||||||||
|
Glycogen. |
||||||||||||||||||
|
Dextrin. |
||||||||||||||||||
|
Cellulose. |
mended. The signs + and — may be conveniently used to indicate positive and negative reaction. Only those carbohydrates which are of greatest importance from the standpoint of physiological chemistry have been included in the chart.
1 Schweitzer's reagent is made by adding potassium hydroxide to a solution of cupric sulphate which contains some ammonium chloride. A precipitate of cupric hydroxide forms and this is filtered off, washed, and 3 grams of the moist cupric hydroxide brought into solution in a liter of 20 per cent ammonium hydroxide.
CARBOHYDRATES.
5'
H
O 8-
|
£ |
r^ |
|
o |
B |
|
t— < |
"c5 |
|
H |
M-< |
|
U |
u |
|
w |
J* |
|
H |
a |
|
W |
B |
|
P |
S-. o |
|
H |
'- E |
|
3.2 |
|
|
tf |
<u ti |
|
O |
c^d |
|
fe |
„ o GJ tf> |
|
w |
.3 <u |
|
"«ri |
*rt .2 |
|
<H |
^*o |
|
w |
rt.2 |
|
ffi |
'/. rH |
|
u |
•" "53 |
|
CO |
§'£ |
|
3 iJ |
|
|
.E-Q |
|
|
o 5 |
|
|
<U TO |
|
|
J3 <u |
fe
|
i! |
3 O C O"? . |
1 U «) ^ |
|
O |
" sol rop d a Co droxi test |
tion absei in 1 " so |
|
own ne d aci nute. . hy. ings |
1 educ the :rose own |
|
|
t/) |
3 O o-p £ *B •3 i- — 3 o |
"-■ CO v» j" |
|
5*2.2 fl/Sh J — i- 2 M |
•^ rt B c ^ Um B O |
|
|
y |
- rtTi o iS >> |
'■5 c: ■£ |
|
B <-; |
the ube vdro "for po st b 1 |
1 <0 »c 0) s 13 ir> rtj c/j — ^ o ^ o F |
|
^1^3, — ° |
S l* 3 I* < |
|
|
2 s |
O W r- *r |
■" xo2o |
|
CJ ^ |
t> CD -TT — |
<U "-"-i tr, 3 |
d «« rt J « rt
"".E B °'rt O
o „ 8 +;£'■§
H 3 B & B —
C q o Cj o
'-2 o JZ E <s>
■pq
a-a-
(* c
3 O
O g
cc 2
|
»3 |
B |
|
a; |
|
|
■<-< to |
u |
|
s |
u> |
|
bos |
rt |
|
Cl B |
2 |
|
<*> £ |
|
|
Gg |
to |
|
^s |
r* |
|
, |
|
|
Ri |
|
|
.§ o |
|
|
C ^ |
TO |
«- cj — 3
._ 4-. I- O ^
fi -3 Cj ^ S
Pi ^ cr. o ~ ^
^ c- rt u «« o
Cj ^n <-m O c^-
|
C cr« |
|
|
B a-> 5 o |
|
|
u ■tt'O |
|
|
o o „ |
|
|
y-° rt^ |
|
|
V4-, 5 O 2 |
|
|
^ 3 be c |
|
|
.5 o o |
z |
I >fl i " I h (!
•^ rt^; .-3 -3 « ^ o ^E-5
- c t3 >>£ S"5 2
5 ^ -3 — ' J. js >-
?3 3 3 o
•3 <u i: ni cj ^J
P ° e ,, oj 3 ° to
£ S •- J •- -B <U
|
.B^S |
g; |
|
|
w to |
'3 o |
|
|
B r* l-< |
co'-^ |
|
|
.2 ^ ° |
o |
|
|
>. rt |
||
|
o"e gj |
^ OJ |
|
|
- 3 ^" |
||
|
^ f,-2 |
tS: |
|
|
u iy v |
Gj |
|
|
f^rt § |
3 O u ?"' «»iB
cj "3'" 2 •& &«
CO ,+i ^ <L>
TO
<»1 <o
J" >- E .3 bO ii ~
ct C C 3 O ra ?
2 >>
i o
5^
|
*3 |
-", |
|
(J |
bO |
|
P-l |
(y.
52 physiological chemistry.
"Unknown" Solutions of Carbohydrates.
At this point the student will be given several " unknown " solu- tions, each solution containing one or more ot the carbohydrates studied. He will be required to detect, by means of the tests on the preceding pages, each carbohydrate constituent of the several "unknown" solutions and hand in, to the instructor, a written report of his findings, on slips furnished by the laboratory.
The scheme given on page 51 may be of use in this connection.
CHAPTER III.
SALIVARY DIGESTION.
The saliva is secreted by three pairs of glands, the submaxillary, sublingual and parotid, reinforced by numerous small glands called buccal glands. The saliva secreted by each pair of glands possesses certain definite characteristics peculiar to itself. For instance, in man, the parotid glands ordinarily secrete a thin, watery fluid, the submaxillary glands secrete a somewhat thicker fluid containing mucin, while the product of the sublingual glands has a more muci- laginous character. The saliva as collected from the mouth is the combined product of all the glands mentioned.
The saliva may be induced to flow by many forms of stimuli, such as chemical, mechanical, electrical, thermal and psychical, the nature and amount of the secretion depending, to a limited degree, upon the particular class of stimuli employed as well as upon the character of the individual stimulus. For example, in experiments upon dogs it has been found that the mechanical stimulus afforded by dropping several pebbles into the animal's mouth caused the flow of but one or two drops of saliva, whereas the mechanical stimulus afforded by sand thrown into the mouth induced a copious flow of a thin watery fluid. Again, when ice-water or snow was placed in the animal's mouth no saliva was seen, while an acid or anything pos- sessing a bitter taste, which the dog wished to reject, caused a free flow of the thin saliva. On the other hand, when articles of food were placed in the dog's mouth the animal secreted a thicker saliva having a higher mucin content — a fluid which would lubricate the food and assist in the passage of the bolus through the oesophagus. It was further found that by simply drawing the attention of the animal to any of the substances named above, results were obtained similar to those secured when the substances were actually placed in the animal's mouth. For example, when a pretense was made of throwing sand into the dog's mouth, a watery saliva was secreted, whereas food under the same conditions excited a thicker and more slimy secretion. The exhibition of dry food, in which the dog had no particular interest (dry bread) caused the secretion of a large
53
54 PHYSIOLOGICAL CHEMISTRY.
amount of watery saliva, while the presentation of moist fooclr which was eagerly desired by the animal, called forth a much smaller secretion, slimy in character. These experiments show it to be rather difficult to differentiate between the influence of physio- logical and psychical stimuli.
The amount of saliva secreted by an adult in 24 hours has been variously placed, as the result of experiment and observation, be- tween 1000 and 1500 c.c, the exact amount depending, among other conditions, upon the character of the food.
The saliva ordinarily has a weak, alkaline reaction to litmus, but becomes acid, in some instances, 2-3 hours after a meal or during fasting. The alkalinity is due principally to di-sodium hydrogen phosphate (Na2HP04) and its average alkalinity may be said to be equivalent to 0.08-0.1 per cent sodium carbonate. The saliva is the most dilute of all the digestive secretions, having an average specific gravity of 1.005 and containing only 0.5 per cent of solid matter. Among the solids- are found albumin, globulin, mucin, urea, the enzymes, salivary amylase (ptyalin) and maltase, phosphates and other inorganic constituents. Potassium thiocyanate, KSCN, is also generally present in the saliva. It. has been claimed that this substance is present in greatest amount in the saliva of habitual smokers. The significance of thiocyanate in the saliva is not known ; it probably comes from the ingested thiocyanates and from the breaking down of protein material.
The so-called tartar formation on the teeth is composed almost entirely of calcium phosphate with some calcium carbonate, mucin, epithelial cells and organic debris derived from the food. The cal- cium salts are held in solution as acid salts, and are probably pre- cipitated by the ammonia of the breath. The various organic sub- stances just mentioned are carried down in the precipitation of the .calcium salts.
The principal enzyme of the saliva is known as salivary amylase or ptyalin. This is an amylolytic enzyme (see p. 3), so-called because it possesses the property of transforming complex carbo- hydrates such as starch and dextrin into simpler bodies. The action of salivary amylase is one of hydrolysis and through this action a series of simpler bodies are formed from the complex starch. The first product of the action of the ptyalin of the saliva upon starch paste is soluble starch (amidulin) and its forma- tion is indicated by the disappearance of the opalescence of the starch solution. This body resembles true starch in giving a
SALIVARY DIGESTION. 55
blue color with iodine. Next follows the formation, in succession, of a series of dextrins, called erythro-dextrin, a-achroo-dcxlrin , (3-achroo-dextrin and. y-achroo-dextrin, the erythro-dextrin being formed directly from soluble starch and later being itself trans- formed into a-achroo-dextriu from which in turn are produced /?- achroo-dextrin and y-achroo-dextrin. Accompanying each dextrin a small amount of iso-maltose is formed, the quantity of iso-mal- tose growing gradually larger as the process of transformation pro- gresses. (Erythro-dextrin gives a red color with iodine, the other dextrins give no color.) The next stage is the transformation of the y-achroo-dextrin into iso-maltose and subsequently the transfor- mation of the iso-maltose into maltose, the latter being the princi- pal end-product of the salivary digestion of starch. At this point a small amount of dextrose is formed from the maltose, through the action of the enzyme maltase.
Salivary amylase acts in alkaline, neutral or combined acid solu- tions. It will act in the presence of relatively strong combined HC1 (see page 119), whereas a trace (0.003 Per cent to 0.006 per cent) of ordinary free hydrochloric acid will not only prevent the action but will destroy the enzyme. By sufficiently increasing the alkalinity of the saliva to litmus, the action of the salivary amylase is inhibited. It has recently been shown by Cannon, that salivary digestion may proceed for a considerable period after the food reaches the stomach, owing to the slowness with which the con- tents are thoroughly mixed with the acid gastric juice and the conse- quent tardy destruction of the enzyme. Food in the pyloric end of the stomach is soon mixed with the gastric secretion but food in the cardiac end is not mixed with the acid gastric juice for a con- siderable period of time and in this region during that time sali- vary digestion may proceed undisturbed.
Maltase, sometimes called glucase, is the second enzyme of the saliva. It is an amylolytic enzyme whose principal function is the splitting of maltose into dextrose. Besides occurring in the saliva it is also present in the pancreatic and intestinal juices. For experi- mental purposes the enzyme is ordinarily prepared from corn. The principles of the " reversibility " of enzyme action were first demonstrated in connection with maltase by Croft Hill.
Microscopical examination of the saliva reveals salivary corpus- cles, bacteria, food debris, epithelial cells, mucus and fungi. In certain pathological conditions of the mouth, pus cells and blood cor- puscles may be found in the saliva.
56
PHYSIOLOGICAL CHEMISTRY.
Experiments on Saliva.
A satisfactory method of obtaining the saliva necessary for the experiments which follow is to chew a small piece of pure paraffin wax, thus stimulating the flow of the secretion, which may be col- lected in a small beaker. Filtered saliva is to be used in every ex- periment except for the microscopical examination.
i. Microscopical Examination. — Examine a drop of unfiltered saliva microscopically and compare with Fig. 18 below.
2. Reaction. — Test the reaction to litmus.
Fig. 18.
^ :%'•
Microscopical Constituents of Saliva.
a, Epithelial cells ; b, salivary corpuscles ; c, fat drops ; d, leucocytes ; e, f and gx bacteria ; h, i and k, fission-fungi.
3. Specific Gravity. — Partially fill a urinometer cylinder with saliva, introduce the urinometer, and observe the reading.
4. Test for Mucin. — To a small amount of saliva in a test-tube add 1-2 drops of dilute acetic acid. Mucin is precipitated.
5. Biuret Test.1 — Render a little saliva alkaline with an equal volume of KOH and add a few drops of a very dilute (2-5 drops in a test-tube of. water) cupric sulphate solution. The formation of a purplish-violet color is due to mucin. .
6. Millon's Reaction.2 — Add a few drops of Millon's reagent to a little saliva. A light yellow precipitate formed by the mucin gradually turns red upon being gently heated.
7. Preparation of Mucin. — Pour 25 c.c. of saliva into 100 c.c. of 95 per cent alcohol, stirring constantly. Cover the vessel and allow the precipitate to stand at least 12 hours. Pour off the supernatant liquid, collect the precipitate on a filter and wash it, in
The significance of this reaction is pointed out on page 92. The significance of this reaction is pointed out on page 90.
SALIVARY DIGESTION. 5/
turn, with alcohol and ether. Finally dry the precipitate, remove it from the paper and make the following tests on the mucin: (a) Test its solubility in the ordinary solvents (see page 23), (b) Millon's reaction, (c) dissolve a small amount in KOH, and try the biuret test on the solution, (d) boil the remainder, with 10-25 c.c. of water to which 5 c.c. of dilute HC1 has been added, until the solution becomes brownish. Cool, render alkaline with solid KOH, and test by Fehling's solution. A reduction should take place. Mucin is what is known as a conjugated protein or glyco- protein (see p. 87) and upon boiling with the acid the carbohydrate group in the molecule has been split off from the protein portion and its presence is indicated by the reduction of Fehling's solution.
8. Inorganic Matter. — Test for chlorides, phosphates, sulphates and calcium. For chlorides, acidify with HN03 and add AgN03. For phosphates, acidify with HN03, heat and add molybdic solu- tion.1 For sulphates, acidify with HC1 and add BaCl2 and warm. For calcium, acidify with acetic acid, CH3COOH, and add ammon- ium oxalate, (NH4)2C204.
9. Viscosity Test. — Place filter papers in two funnels, and to each add an equal quantity of starch paste (5 c.c). Add a few drops of saliva to one lot of paste and an equivalent amount of water to the other. Note the progress of filtration in each case. Why does one solution filter more rapidly than the other?
10. Test for Nitrites. — Add 1-2 drops of dilute H2S04 to a little saliva and thoroughly stir. Now add a few drops of a potas- sium iodide solution and some starch paste. Nitrous acid is formed which liberates iodine, causing the formation of the blue iodide of starch.
11. Thiocyanate Tests. — (a) Ferric Chloride Test, — To a little saliva in a small porcelain crucible, or dish, add a few drops of dilute ferric chloride and acidify slightly with HC1. Red ferric thiocyanate forms. To show that the red coloration is not due to iron phosphate add a drop of HgCl2 when colorless mercuric thio- cyanate forms.
(b) Solera's Reaction. — This test depends upon the liberation of iodine through the action of thiocyanate upon iodic acid. Moisten
1 Molybdic solution is prepared as follows, the parts being by weight :
1 part, molybdic acid.
4 parts, ammonium hydroxide (Sp. gr. 0.96). 15 parts, nitric acid (Sp. gr. 1.2).
58 PHYSIOLOGICAL CHEMISTRY.
a strip of starch paste-iodic acid test paper1 with a little saliva. If thiocyanate be present the test paper will assume a blue color, due to the liberation of iodine and the subsequent formation of the so-called iodide of starch.
12. Digestion of Starch Paste. — To 25 c.c. of starch paste in a small beaker, add 5 drops of saliva and stir thoroughly. At in- tervals of a minute remove a drop of the solution to one of the de- pressions in a test-tablet and test by the iodine test. If the blue color with iodine still forms after 5 minutes, add another 5 drops of saliva. The opalescence of the starch solution should soon dis- appear, indicating the formation of soluble starch which gives a blue color with iodine. This body should soon be transformed into erythro-dextrin which gives a red color with iodine and this in turn should pass into achroo-dextrin which gives no color with iodine. This is called the achromic point. When this point is reached test by Fehling's test to show the production of a reducing body. A positive Fehling's test may be obtained while the solution still reacts red with iodine inasmuch as some iso-maltose is formed from the soluble starch coincidently with the formation of the erythro-dextrin. How long did it take for a complete transforma- tion of the starch?
13. Digestion of Dry Starch. — In a test-tube shake up a small amount of dry starch with a little water. Add a few drops of saliva, mix well and allow to stand. After 10-20 minutes filter and test the filtrate by Fehling's test. What is the result and why ?
14. Digestion of Inulin. — To 5 c.c. of inulin solution in a test- tube add 10 drops of saliva and place the tube in the water-bath at 400 C. After one-half hour test the solution by Fehling's test.2 Is any reducing substance present ? What do you conclude regard- ing the salivary digestion of inulin?
15. Influence of Temperature. — In each of four tubes place about 5 c.c. of starch paste. Immerse one tube in cold water from the faucet, keep a second at room temperature and place a third on the water-bath at 40 ° C. Now add to the contents of each of these three tubes two drops of saliva and shake well; to the
1 This test paper is prepared as follows : Saturate a good quality of filter paper with 0.5 per cent starch paste to which has been added sufficient iodic acid to make a 1 per cent solution of iodic acid and allow the paper to dry in the air. Cut it in strips of suitable size and preserve for use.
2 If the inulin solution gives a reduction before being acted upon by the saliva it will be necessary to determine the extent of the original reduction by means of a "check" test (see page 47).
SALIVARY DIGESTION. 59
contents of the fourth tube add two drops of boiled saliva. Test frequently by the iodine test, using the test-tablet, and note in which tube the most rapid digestion occurs. Explain the results.
16. Influence of Dilution. — Take a series of 6 test-tubes each containing 9 c.c. of water. Add 1 c.c. of saliva to tube 1 and shake thoroughly. Remove 1 c.c. of the solution from tube 1 to tube 2 and after mixing' thoroughly remove 1 c.c. from tube 2 to tube 3. Continue in this manner until you have 6 saliva solutions of gradu- ally decreasing strength. Now add starch paste in equal amounts to each tube, mix very thoroughly and place on the water-bath at 400 C. After 10-20 minutes test by both the iodine and Feh- ling's tests. In how great dilution does your saliva act?
17. Influence of Acids and Alkalis. — (a) Influence of Free Acid. — Prepare a series of six tubes in each of which is placed 4 c.c. of one of the following strengths of free HC1 : 0.2 per cent, o. 1 per cent, 0.05 per cent, 0.025 per cent, 0.0125 per cent and 0.006 per cent. Now add 2 c.c. of starch paste to each tube and shake them thoroughly. Complete the solutions by adding 2 c.c. of saliva to each and repeat the shaking. The total acidity of this series would be as follows: 0.1 per cent, 0.05 per cent, 0.025 per cent, 0.0125 per cent, 0.006 per cent and 0.003 Per cent- Place these tubes on the water-bath at 40° C. for 10-20 minutes. Divide the contents of each tube into two parts, testing one part by the iodine test and testing the other, after neutralization, by Fehling's test. What do you find?
(b) Influence of Combined Acid. — Repeat the first three experi- ments of the above series using combined hydrochloric acid (see page 119) instead of the free acid. How does the action of the combined acid differ from that of the free acid?
(c) Influence of Alkali. — Repeat the first four experiments under (a) replacing the HC1 by 2 per cent, 1 per cent, 0.5 per cent and 0.25 per cent Na2C03. Neutralize the alkalinity before trying the iodine test (see Starch, 5, page 44).
(d) Nature of the Action of Acid and Alkali. — Place 2 c.c. of saliva and 2 c.c. of 0.2 per cent HC1 in a test-tube and leave for 15 minutes. Neutralize the solution, add 4 c.c. of starch paste and place the tube on the water-bath at 400 C. In 10 minutes test by the iodine and Fehling's tests and explain the result. Repeat the experiment, replacing the 0.2 per cent HC1 by 2 per cent Na2C03. What do you deduce from these two experiments?
18. Influence of Metallic Salts, etc. — In each of a series of
60 PHYSIOLOGICAL CHEMISTRY.
tubes place 4 c.c. of starch paste and x/z c.c. of one of the solutions named below. Shake well, add ^ c.c. of saliva to each tube, thor- oughly mix, and place on the water-bath at 400 C. for 10-20 minutes. Show the progress of digestion by means of the iodine and Fehling tests. Use the following chemicals : Metallic salts, 10 per cent plumbic acetate, 2 per cent cupric sulphate, 5 per cent ferric chloride, 8 per cent mercuric chloride; Neutral salts, 10 per cent sodium chloride, 10 per cent magnesium sulphate, 3 per cent barium chloride, 10 per cent Rochelle salt. Also try the influence of 2 per cent carbolic acid, 95 per cent alcohol, and ether and chlor- oform. What are your conclusions?
19. Excretion of Potassium Iodide. — Ingest a small dose of potassium iodide (0.2 gram) contained in a gelatin capsule, quickly rinse out the mouth with water and then test the saliva at once for iodine. This test should be negative. Make additional tests for iodine at 2 minute intervals. The test for iodine is made as fol- lows : Take 1 c.c. of NaN02 and 1 c.c. of dilute HoSCV in a test- tube, add a little saliva directly from the mouth, and a small amount of starch paste. If convenient, the urine may also be tested. The formation of a blue color signifies that the potassium iodide is being excreted through the salivary glands. Note the length of time elapsing between the ingestion of the potassium iodide and the appearance of the first traces of the substance in the saliva. The chemical reactions taking place in this experiment are indicated in the following equations :
( a) 2NaN02 + H2S04 = 2HN02< + Na2S04.
( b ) 2KI + H2S04 = 2HI + K2S04.
(c) 2HN02 + 2HI = I2 + 2H20 + 2NO.
20. Qualitative Analysis of the Products of Salivary Diges- tion.— To 25 c.c. of the products of salivary digestion (saved from Experiment 12 or furnished by the instructor), add 100 c.c. of 95 per cent alcohol. Allow to stand until the white precipitate has settled. Filter, evaporate the filtrate to dryness, dissolve the resi- due in 5-10 c.c. of water and try Fehling's test (page 27) and the phenylhydrazine reaction (see Dextrose, 3, page 24). On the dex- trin precipitate try the iodine test (page 44). Also hydrolyze the dextrin as given under Dextrin, 4, page 48.
1 Instead of this mixture a few drops of HNOs possessing a yellowish or brownish color due to the presence of HNO2 may be employed.
CHAPTER IV.
PROTEINS.1 THEIR DECOMPOSITION AND SYNTHESIS.
The proteins are a class of substances, which in the light of our present knowledge, consist, in the main of combinations of a- amino-acids or their derivatives. These protein substances form the chief constituents of many of the fluids of the body, constitute the organic basis of animal tissue, and at the same time occupy a decidedly preeminent position among our organic food-stuffs. They are absolutely necessary to the uses of the animal organism for the continuance of life and they cannot be satisfactorily replaced in the diet of such an organism by any other dietary constituent either organic or inorganic. Such an organism may exist without pro- tein food for a period of time, the length of the period varying according to the specific organism and the nature of the substitu- tion offered for the protein portion of the diet. Such a period is, however, distinctly one of existence rather than one of normal life and one which is consequently not accompanied by such a full and free exercise of the various functions of the organism as would be possible upon an evenly balanced ration, i. c., one containing the requisite amount of protein food. These protein substances, are. furthermore, essential constituents of all living cells and therefore without them vegetable life as well as animal life is impossible.
The proteins, which constitute such an important group of sub- stances, differ from the carbohydrates and fats very decidedly in elementary composition. In addition to containing carbon, hy- drogen, and oxygen, which are present in fats and carbohydrates, the proteins invariably contain nitrogen in their molecule and gen- erally sulphur also. Proteins have also been identified which con- tain phosphorus, iron, copper, iodine, manganese, and sine. The percentage composition of the more important members of the group of protein substances would fall within the following limits : C = 50-55 per cent, H= 6-7.3 Per cent- 0 = 19-24 per cent, N= 15-19 per cent. 5 = 0.3-2.5 per cent, P = 0.4-0.8 per cent
1 The term proteid has been very widely used by English-speaking scientists to signify the class of substances we have called proteins.
61
62 PHYSIOLOGICAL CHEMISTRY.
when present. When iron, copper, iodine, manganese, or zinc are present in the protein molecule they are practically without excep- tion present only in traces}
Of all the various elements of the protein molecule, nitrogen is by far the most important. The human body needs nitrogen for the continuation of life, but it cannot use the nitrogen of the air or that in various other combinations as we find it in nitrates, nitrites, etc. However, in the protein molecule the nitrogen is pres- ent in a form which is utilizable by the body. The nitrogen in the protein molecule occurs in at least four different forms as follows :
I. Monamino acid nitrogen. II. Diamino acid nitrogen or basic nitrogen.
III. Amide nitrogen.
IV. A guanidine residue.
The actual structure of the protein molecule is still unknown, and we have as yet no means by which its molecular weight can be even approximately established. The many attempts which have been made to determine this have led to very different results, some of which are given in the following table:
Serum albumin = 4572-5 100-5 135 Egg albumin = 4900-6542 Globin = 1 5000-16086
Oxyhemoglobin = 1 4800-1 5000-1 665 5-1 6730
Of these figures, those given for oxyhemoglobin deserve the most consideration, for these are based on the atomic ratios of the sulphur and iron contained in this substance. The simplest formula that can be calculated from analyses of oxyhemoglobin, namely, C658H1181N207S2FeO210, serves to show the great complexity of this substance. The following formulas which have been proposed for typical protein substances may serve to further impress the fact of the great size of the protein molecule :
Egg albumin = C239H386N5SS207S Serum albumin = C450H720N11GS6O140
The decomposition2 of protein substances may be brought about
1 Some investigators regard these elements as contaminations, or constituents of some non-protein substance combined with the protein.
2 The terms " degradation," " dissociation," and " cleavage," are often used in this connection.
PROTEINS.
63
by oxidation or hydrolysis, but inasmuch as the hydrolytic proce- dure has been productive of the more satisfactory results, that type of decomposition procedure alone is used at present. This hydrolysis of the protein molecule may be accomplished by acids, alkalis, or superheated steam, and in digestion by the action of the proteolytic enzymes. The character of the decomposition products varies ac- cording to the method utilized in tearing the molecule apart. Bear- ing this in mind, we may say that the decomposition products of proteins include proteoses, peptones, peptides, carbon dioxide, am- monia, hydrogen sulphide, and amino acids. These amino acids constitute a long list of important substances which contain nuclei belonging either to the aliphatic, carbocyclic, or heterocyclic series. The list includes, glycocoll, alanine, serine, phenylalanine, tyrosine, cystine, tryptophane, histidine, valine, arginine, leucine, isoleucine, lysine, aspartic acid, glutamic acid, proline, oxy proline, and diamino- trihydroxydodecanoic acid. Of these amino acids, tyrosine and phenylalanine contain carbocyclic nuclei, histidine, proline and tryp- tophane contain heterocyclic nuclei, and the remaining members of the list, as given, contain aliphatic nuclei. The amino acids are pre- eminently the most important class of protein decomposition prod- ucts. These amino acids are all a-amino acids, and, with the exception of glycocoll, are all optically active. Furthermore they are amphoteric substances and consequently are able to form salts with both bases and acids. These properties are inherent in the NH2 and COOH groups of the amino acids.
The decomposition products of protein may be grouped as pri- mary and secondary decomposition products. By primary products are meant those which exist as radicals within the protein molecule and which are liberated, upon cleavage of this molecule, with their carbon chains intact and the position of their nitrogen unaltered. The secondary products are those which result from the disintegra- tion of the primary cleavage products. No matter what method is used to decompose a given protein molecule, the primary products are largely the same under all conditions.1
In the process of hydrolysis the protein molecule is gradually broken dow.n and less complicated aggregates than the original molecule are formed, which are known as proteoses, peptones and peptides and which still possess true protein characteristics. Fur- ther hydrolysis causes the ultimate transformation of these sub-
1 Alkaline hydrolysis yields urea and ornithine which result from arginine, the product of acid hydrolysis.
64 PHYSIOLOGICAL CHEMISTRY.
stances, of a protein nature, into the amino acids of known chemi- cal structure. In this decomposition the protein molecule is not broken down in a regular manner into y2, Ya, % portions and the amino acids formed in a group at the termination of the hy- drolysis. On the contrary, certain amino acids are formed very early in the process, in fact while the main hydrolytic action has proceeded no further than the proteose stage. Gradually the com- plexity of the protein portion undergoing decomposition is sim- plified by the splitting off of the amino acids and finally it is so far decomposed through previous cleavages that it yields only amino acids at the succeeding cleavage. In short the general plan of the hydrolysis of the protein molecule is similar to the hydrolysis of starch. In the case of starch there is formed a series of dextrins of gradually decreasing complexity and coincidently with the formation of each dextrin a small amount of sugar is split off and finally nothing but sugar remains. In the case of protein hydrolysis there is a series of proteins of gradually decreasing complexity produced and coincidently with the formation of each new protein substance amino acids are split off and finally the sole products remaining are amino acids.
Inasmuch as diversity in the method of decomposing a given protein does not result in an equally diversified line of decomposition products, but, on the other hand, yields products which are quite comparable in character, it may be argued that there are probably well denned lines of cleavage in the individual protein molecule and that no matter what the force brought to bear to tear such a molecule apart, the disintegration, when it comes, will yield in every case, certain definite fragments. These fragments may be called the " building stones " of the protein molecule, a term used by some of the German investigators. Take, for example, the decomposition of protein which may be brought about through the action of the enzyme trypsin of the pancreatic juice. When this enzyme is allowed to act upon a given protein, the latter is disintegrated in a series of definite cleavages, resulting in the for- mation of proteoses, peptones and peptides in regular order, the peptides being the last of the decomposition products which possess protein characteristics. They are all built up from amino acids and are therefore closely related to these acids on the one side and to peptones on the other. We have di-, tri-, tetra-, penta-, deca-, and poly-peptides which are named according to the number of amino acids included in the peptide molecule. Following the peptides
PROTEINS. 65
there are a diverse assortment of monamino and diamino acids which constitute the final products of the protein decomposition. These acids are devoid of any protein characteristics and are there- fore decidedly different from the original substance from which they were derived. From a protein of huge molecular weight, a typical colloid, perhaps but slightly soluble, and entirely non-dif- fusible, we have passed by way of proteoses, peptones, and pep- tides to a class of simpler crystalline substances which are, for the most part, readily soluble and diffusible.
These amino acids after their production in the process of diges- tion, as just indicated, are synthesized within the organism to form protein material which goes to build up the tissues of the body. It is thus seen that the amino acids are of prime importance in the animal economy. Moreover, it is important to remember that these essential factors in metabolism and nutrition cannot be produced within the animal organism from their elements, but are only yielded upon the hydrolysis of ingested protein of animal or vegetable origin.
When we examine the formulas of the principal members of the crystalline end-products of protein decomposition we note that they are invariably acids, as has already been mentioned, and contain an NH2 group in the a position. This relation of the NH2 group to the acid radical is constant, no matter what other groups or radicals are present. We may have straight chains as in alanine and glu- tamic acid, the benzene ring as in phenylalanine or we may have sulphurised bodies as in cystine and still the formula is always of the same type, i. e.,
NH2
I E - CH - COOH
It is seen that this characteristic grouping in the amino acid pro- vides each one of these ultimate fragments of the protein molecule with both a strong acid and a strong basic group. For this reason it is theoretically possible for a large number of these amino acids to combine and the resulting combinations may be very great in number, since there is such a varied assortment of the acids. The protein molecule, which is of such mammoth proportions, is prob- ably constructed on a foundation of this sort. Of late much valu- able data have been collected regarding the synthetic production of protein substances, the leaders in this line of investigation being Fischer and Abderhalden. After having gathered a mass of data 6
66 PHYSIOLOGICAL CHEMISTRY.
regarding the final products of the protein decomposition and demonstrating that amino acids were the ultimate results of the various forms of decomposition, these investigators, and notably Fischer, set about in an effort to form, from these amino acids, by- synthetic means, substances which should possess protein character- istics. The simplest of these bodies formed in this way was synthe- sized from two molecules of glycocoll with the liberation of water, thus:
CH9 - NEL - CO iOH Hi HN - CEL - COOH.
L2 -L-N J.O-2
The body thus formed is a dipeptide, called glycyl-glycine. In an analogous manner may be produced leucyl-leucine , through the synthesis of two molecules of leucine or leucyl-alanyl- glycine through the union of one molecule of leucine, one of alanine, and one of glycocoll. By this procedure Fischer and his pupils have been able to make a large number of peptides containing varied numbers of amino acid radicals, the name polypeptides being given to the whole group of synthetic substances thus formed. The most complex poly- peptide yet produced is one containing fifteen glycocoll and three leucine residues.
Notwithstanding the fact that most synthetic polypeptides are produced through a union of amino acids by means of their imide bonds, it must not be imagined that the protein molecule is con- structed from amino acids linked together in straight chains in a manner analogous to the formation of simple peptides, such as glycyl-glycine. The molecular structure of the proteins is much too complex to be explained upon any such simple formation as that. There must be a variety of linkings, since there is a varied assort- ment of decomposition products of totally different structure.
Many of these synthetic bodies respond to the biuret test, are precipitated by phosphotungstic acid and behave, in other ways, as to leave no doubt as to their protein characteristics. For instance, a number of amino acids each possessing a sweet taste have been synthesized in such a manner as to yield a polypeptide of bitter taste, a well known characteristic of peptones. From the fact that the polypeptides formed in the manner indicated have free acidic and basic radicals we gather the explanation of the amphoteric character of true proteins. Fischer expresses the encouraging belief that he will soon be able to produce a true protein by the synthesis of its decomposition products. Silk fibroin is the protein substance he expects to synthesize. He no doubt will perform this
PROTEINS. 67
joint office for organic and physiological chemistry if it is capable of performance by the present methods of technique. Even Fischer, however, is frank enough to say that the production of the great body of protein substances synthetically, will, under the most en- couraging conditions, be a terrific task, involving the " life-work of a whole army of inventive and diligent chemists."
For the benefit of those especially interested in such matters a photograph of the Fischer apparatus (Fig. 22, page 71) used in the fractional distillation, in vacuo, of the esters of the decompo- sition products of the proteins, as well as micro-photographs and drawings of preparations of several of these decomposition products (Figs 19 to 31, pages 68 to 81) are introduced. For the prepara- tions and the photograph of the apparatus the author is indebted to Dr. T. B. Osborne, of New Haven, Conn., who has made many important observations upon the hydrolysis of proteins. The repro- duction of the crystalline form of some of the more recent of the products may be of interest to those viewing the field of physio- logical chemistry from other than the student's aspect.
An extended discussion of the various decomposition products being out of place in a book of this character, we will simply make a few general statements in connection with the primary decompo- sition products.
DISCUSSION OF THE PRODUCTS.
Ammonia, NH3. — Ammonia is an important decomposition product of all proteins and probably arises from an amide group combined with a carboxyl group of some of the amino acids. It is possible that the dibasic acids, aspartic and glutamic, furnish most of these carboxyl groups. This is indicated by the more or less close relationship which exists between the amount of ammonia and that of the dibasic acids which the several proteins yield upon decomposition. The elimination of the ammonia from proteins under the action of acids and alkalis is very similar to that from amides like asparagine.
Glycocoll, C2H5N02. — Glycocoll, or amino acetic acid, is the simplest of the amino acids and has the following formula :
NH2
H-C-COOH.
I H
68
PHYSIOLOGICAL CHEMISTRY.
Glycocoll, as the formula shows, contains no asymmetric carbon atom, and is the only amino acid yielded by protein decomposition which is optically inactive. Glycocoll and leucine were the first decom- position products of proteins to be discovered (1820). Upon ad- ministering benzoic acid to animals the output of hippuric acid in the urine is greatly increased, thus showing a synthesis of benzoic acid and glycocoll in the organism (see p. 160, Chapter IX) . Glyco- coll, ingested in small amount, is excreted in the urine as urea, whereas if administered in excess it appears in part unchanged in the urine. It is usually separated from the mixture of protein de- composition products as the hydrochloride of the ester. The crys- talline form of this compound is shown in Fig. 19.
Fig. 19.
Glycocoll Ester Hydrochloride.
Alanine, C3H7N02. — Alanine is a-amino-propionic acid, and as such it may be represented structurally as follows :
H NH2
H-C-C-COOH.
H H
Obtained from protein substances, alanine is dextro-rotatory, is very soluble in water, and possesses a sweet taste. Tyrosine, phenylalanine, cystine and serine are derivatives of alanine. This amino acid has been obtained from nearly all proteins examined. Its absence, from those proteins from which it has not been obtained.
PROTEINS.
69
has not been proven. Most proteins yield relatively small amounts of alanine.
Serine, C3H7N03. — Serine is a-amino-P-hydroxy-propionic acid and possesses the following structural formula :
OH NH2
H _ C - C - COOH
I I H H
Fig. 20.
Serine.
Serine obtained from proteins is laevo-rotatory, possesses a sweet taste and is quite soluble in water. Serine is not obtained in quantity from most proteins but is yielded abundantly by silk glue. Owing to the difficulty of separating serine it has not been found in a number of proteins in which it probably occurs. Serine crystals are shown in Fig. 20, above.
Phenylalanine, C9HuN02. — This product is phenyl-a-amino- propionic acid, and may be represented graphically as follows :
H NHo
-C-C-COOH. H H
The laevo-rotatory form is obtained from proteins. Phenylalanine has been obtained from all the proteins examined except from the
yo
PHYSIOLOGICAL CHEMISTRY.
protamines and some of the albuminoids. The yield of this body from the decomposition of proteins is frequently greater than the
Fin. 21.
Phenylalanine.
yield of tyrosine. The crystalline form of phenylalanine is shown in Fig. 21.
Tyrosine, CgH^NC^. — Tyrosine, one of the first discovered end- products of protein decomposition, is the amino acid, p-oxyphenyl- a-amino-propionic acid. It has the following formula :
H NH2
I I _C-C-COOH.
H H
OH
The tyrosine which results from protein decomposition is usually lsevo-rotatory although the dextro-rotatory form sometimes occurs. Tyrosine is one of the end-products of tryptic digestion and usually separates in conspicuous amount early in the process of digestion. It does not occur, however, as an end-product of the decomposition of gelatin.
Tyrosine is found in old cheese, and derives its name from this fact. It crystallizes in tufts, sheaves or balls of fine needles, which decom- pose at 295 ° C. and are sparingly soluble in cold (1-2454) water,
PROTEINS.
7<
but much more so in boiling (1-154) water. Tyrosine forms sol- uble salts with alkalis, ammonia or mineral acids, and is soluble,
Fig. 22.
Fischer Apparatus.
Reproduced from a photograph made by Prof. E. T. Reichert, of the University of Pennsylvania. The negative was furnished by Dr. T. B. Osborne, of New Haven, Conn.
A, Tank into which freezing mixture is pumped and from which it flows through the condenser, B ; C, flask from which the esters are distilled, the distillate being collected in D ; E, a Dewar flask containing liquid air serving as a cooler for con- densing tube F ; G and G' , tubes leading to the Geryck pump by which the vacuum is maintained ; /, tube leading to a McLeod gauge (not shown in figure) ; /, a bath con- taining freezing mixture in which the receiver D is immersed ; K, a bath of water during the first part of the distillation and of oil during the last part of the process ; 1-5, stop cocks which permit the cutting out of different parts of the apparatus as the procedure demands.
with difficulty, in acetic acid. It responds to Millon's reaction, thus showing the presence of the hydroxyphenyl group, but gives no
72
PHYSIOLOGICAL CHEMISTRY.
other protein test. The aromatic groups present in tyrosine, phenyl- alanine and tryptophane cause proteins to yield a positive xantho- proteic reaction. In severe cases of typhoid fever and smallpox,
Fig.
23-
Tyrosine.
in acute yellow atrophy of the liver, and in acute phosphorus poison- ing-, tyrosine has been found in the urine. Tyrosine crystals are shown in Fig. 23, above.
Cystine, C6H1204N2S2. — Friedmann has recently shown cystine to be the disulphide of a-amino-fi-thiolactic acid1 and to possess the following structural formula:
CH2-S — kvCxl2
CHNH2 CH-NH2
I I
COOH COOH.
Cystine is the principal sulphur-containing body obtained from the decomposition of protein substances. It is obtained in greatest amount as a decomposition product of such keratin-containing tis- sues as horn, hoof and hair. Cystine occurs in small amount in normal urine and is greatly increased in quantity under certain pathological conditions. It crystallizes in thin, colorless hexagonal plates which are shown in Fig. 24, p. 73. Cystine is very slightly soluble in water but its salts, with both bases and acids, are readily soluble in water. It is lsevo-rotatory.
1 Also called a-diamino-/3-dithio-dilactylic acid.
PROTEINS. 73
It has recently been claimed that cystine occurs in two forms, i. e., stone-cystine and protein-cystine and that these two forms are distinct in their properties. This view is incorrect.
Fig. 2a.
Cystine.
For a discussion of cystine sediments in urine see Chapter XX.
Tryptophane, CnH12N202. — According to Ellinger, tryptophane is indol-amino-propionic acid. Recently Ellinger and Flamand have shown that it possesses the following formula :
/\ C-CH2CH(NH2)-COOH
U\Ah
NH
Tryptophane is the mother-substance of indole, skatole, skatole acetic acid and skatole carboxylic acid, all of which are formed as secondary decomposition products of proteins. Its presence in protein substances may be shown by means of the Adamkiewicz reaction or the Hopkins-Cole reaction (see page 91). It may be detected in a tryptic digestion mixture through its property of giving a violet color-reaction with bromine water. Tryptophane is yielded by nearly all proteins but has been shown to be entirely absent from zein, the prolamin (alcohol-soluble protein) of maize.
Solutions of tryptophane in sodium hydroxide are dextro-rotatory. Upon being heated to 266 ° C. tryptophane decomposes with the evo- lution of gas.
72
PHYSIOLOGICAL CHEMISTRY.
other protein test. The aromatic groups present in tyrosine, phenyl- alanine and tryptophane cause proteins to yield a positive xantho- proteic reaction. In severe cases of typhoid fever and smallpox,
Fig. 23.
Tyrosine.
in acute yellow atrophy of the liver, and in acute phosphorus poison- ing, tyrosine has been found in the urine. Tyrosine crystals are shown in Fig. 23, above.
Cystine, C6H1204N2S2. — Friedmann has recently shown cystine to be the disulphide of a-amino-P-thiolactic acid1 and to possess the following structural formula :
CH2-S-SCH2
CH-NH2 CH-NH2
I I
COOH COOH.
Cystine is the principal sulphur-containing body obtained from the decomposition of protein substances. It is obtained in greatest amount as a decomposition product of such keratin-containing tis- sues as horn, hoof and hair. Cystine occurs in small amount in normal urine and is greatly increased in quantity under certain pathological conditions. It crystallizes in thin, colorless hexagonal plates which are shown in Fig. 24, p. 73. Cystine is very slightly soluble in water but its salts, with both bases and acids, are readily soluble in water. It is lsevo-rotatory.
1 Also called a-diamino-/3-dithio-dilactylic acid.
■"'
PROTEINS.
73
It has recently been claimed that cystine occurs in two forms, i. e., stone-cystine and protein-cystine and that these two forms are distinct in their properties. This view is incorrect.
Fig. 24.
Cystine.
For a discussion of cystine sediments in urine see Chapter XX.
Tryptophane, C^H^^C^. — According to Ellinger, tryptophane is indol-amino-propionic acid. Recently Ellinger and Flamand have shown that it possesses the following formula :
/\ C-CH2CH(NH2)COOH
kAJcH NH
Tryptophane is the mother-substance of indole, skatole, skatole acetic acid and skatole carboxylic acid, all of which are formed as secondary decomposition products of proteins. Its presence in protein substances may be shown by means of the Adamkiewicz reaction or the Hopkins-Cole reaction (see page 91). It may be detected in a tryptic digestion mixture through its property of giving a violet color-reaction with bromine water. Tryptophane is yielded by nearly all proteins but has been shown to be entirely absent from zein, the prolamin (alcohol-soluble protein) of maize.
Solutions of tryptophane in sodium hydroxide are dextro-rotatory. Upon being heated to 266 ° C. tryptophane decomposes with the evo- lution of gas.
72
PHYSIOLOGICAL CHEMISTRY.
other protein test. The aromatic groups present in tyrosine, phenyl- alanine and tryptophane cause proteins to yield a positive xantho- proteic reaction. In severe cases of typhoid fever and smallpox,
Fig. 23.
Tyrosine.
in acute yellow atrophy of the liver, and in acute phosphorus poison- ing, tyrosine has been found in the urine. Tyrosine crystals are shown in Fig. 23, above.
Cystine, C6H1204N2S2. — Friedmann has recently shown cystine to be the disulphide of a-amino-P-thiolactic acid1 and to possess the following structural formula:
CH2* fe — k5*Oxi2
CHNH2 CHNH2
I I
COOH COOH.
Cystine is the principal sulphur-containing body obtained from the decomposition of protein substances. It is obtained in greatest amount as a decomposition product of such keratin-containing tis- sues as horn, hoof and hair. Cystine occurs in small amount in normal urine and is greatly increased in quantity under certain pathological conditions. It crystallizes in thin, colorless hexagonal plates which are shown in Fig. 24, p. 73. Cystine is very slightly soluble in water but its salts, with both bases and acids, are readily soluble in water. It is lsevo-rotatory.
1 Also called a-diamino-/3-dithio-dilactylic acid.
PROTEINS.
73
-
■
s ysfirie
itaining tis-
m
are rw»
•ih
It has recently been claimed that cystine occurs in two forms, i. e., stone-cystine and protein-cystine and that these two forms are distinct in their properties. This view is incorrect.
Fig. 24.
Cystine.
For a discussion of cystine sediments in urine see Chapter XX.
Tryptophane, C1]LH12N202. — According to Ellinger, tryptophane is indol-amino-propionic acid. Recently Ellinger and Flamand have shown that it possesses the following formula:
/\ C-CH2CH(NH2)COOH
NH
Tryptophane is the mother-substance of indole, skatole, skatole acetic acid and skatole carboxylic acid, all of which are formed as secondary decomposition products of proteins. Its presence in protein substances may be shown by means of the Adamkiewicz reaction or the Hopkins-Cole reaction (see page 91). It may be detected in a tryptic digestion mixture through its property of giving a violet color-reaction with bromine water. Tryptophane is yielded by nearly all proteins but has been shown to be entirely absent from zein, the prolamin (alcohol-soluble protein) of maize.
Solutions of tryptophane in sodium hydroxide are dextro-rotatory. Upon being heated to 266 ° C. tryptophane decomposes with the evo- lution of gas.
74
PHYSIOLOGICAL CHEMISTRY.
Histidine, C6H9N302. — Histidine is a-amino-fS-imidazol-pro- pionic acid with the following structural formula :
H NH2
HC = C-C-C-COOH.
I I H H
HN\/N
CH
The histidine obtained from proteins is laevo-rotatory. It has been obtained from all the proteins thus far examined, the majority of them yielding about 2.5 per cent of the amino acid. How- Fig. 25.
Histidine Dichloride.
ever, about 1 1 per cent was obtained by Abderhalden from globin, the protein constituent of oxyhemoglobin and about 13 per cent by Kossel and Kutscher from the protamine sturine.
Crystals of histidine dichloride are shown in Fig. 25, above.
Knoop's Color Reaction for Histidine. — To an aqueous solu- tion of histidine or a histidine salt in a test-tube add a little bromine water. A yellow coloration develops in the cold and upon further addition of bromine water becomes permanent. If the tube be heated,1 the color will disappear and will shortly be replaced by a faint red coloration which gradually passes into a deep wine red. Usually black, amorphous particles separate out and the solution becomes turbid.
1 The same reaction will take place in the cold more slowly.
PROTEINS. 75
The reaction cannot be obtained in solutions containing free alkali. It is best to use such an amount of bromine as will produce a permanent yellow color in the cold. The use of a less amount of bromine than this produces a weak coloration whereas an excess of bromine prevents the reaction. The test is not very delicate, but a characteristic reaction may always be obtained in I : iooo solu- tions. The only histidine derivative which yields a similar colora- tion is imidazolethylamine, and the reaction in this case is rather weak as compared with the color obtained with histidine or histi- dine salts.
Valine, C5HnN02. — The amino-valerianic acid obtained from proteins is a-amino-isovalerianic acid, and as such bears the follow- ing formula :
CH3 NH2
H-C C-COOH.
I I
CH3 H
It closely resembles leucine in many of its properties, but is more soluble in water. It is a difficult matter to identify valine in the presence of leucine and isoleucine inasmuch as these amino acids crystallize together in such a way that the combination persists even after repeated recrystallizations. Valine is dextro-rotatory.
Arginine, C6H14N402. — Arginine is guanidine-a-amino-valeri- anic acid and possesses the following structural formula :
H H H NH2
NH-C-C-C-C-COOH.
I I I I I NH = C H H H H
NH2
It has been obtained from every protein so far subjected to decom- position. The arginine obtained from proteins is dextro-rotatory, and has pronounced basic properties, reacts strongly alkaline to litmus, and forms stable carbonates. Because of these facts, some investigators consider arginine to be the nucleus of the protein molecule. It is obtained in widely different amounts from different proteins, over 85 percent of certain protamines having been obtained in the form of this amino acid. It is claimed that in the ordinary
76
PHYSIOLOGICAL CHEMISTRY.
metabolic activities of the animal body arginine gives rise to urea. While this claim is probably true, it should, at the same time, be borne in mind that the greater part of the protein nitrogen is eliminated as urea and that, therefore, but a very small part can arise from arginine.
Leucine, C6H13N02. — Leucine is an abundant end-product of the decomposition of protein material, and, together with glycocoll, was the first of these products to be discovered (1820). It is a-amino-isobutyl-acetic acid, and therefore has the following for- mula :
CIL
NIL
CH-CH.-C-COOH.
CIL
H
The leucine which results from protein decomposition is /-leucine. Leucine is present normally in the pancreas, thymus, thyroid, spleen, brain, liver, kidneys and salivary glands. It has been found patho- logically in the urine (in acute yellow atrophy of the liver, in acute phosphorus poisoning and in severe cases of typhoid fever and smallpox), and in the liver, blood and pus.
Fig. 26.
Leucine.
Pure leucine crystallizes in thin, white hexagonal plates. Crystals of pure leucine are reproduced in Fig. 26. It is rather easily soluble in water (46 parts), alkalis, ammonia and acids. On rapid heating
PROTEINS. 77
to 2950 C, leucine decomposes with the formation of carbon dioxide, ammonia and amylamine. Aqueous solutions of leucine obtained from proteins are lsevo-rotatory, but its acid or alkaline, solutions are dextro-rotatory. So-called impure leucine1 is a slightly refractive substance, which generally crystallizes in balls having a radial structure or in aggregations of spherical bodies, Fig. 104, Chapter XX.
Isoleucine, CGH13N02. — Isoleucine is a-amino-methyl-ethyl-pro- pionic acid, and possesses the following structural formula:
CH3 NH2 H • C-COOH.
A
2H5 H
This amino acid was recently discovered by Ehrlich. Its presence has been established among the decomposition products of only a few proteins although it probably occurs among those of many or most of them. Ehrlich has shown that the d-amyl alcohol which is produced by yeast fermentation originates from isoleucine and the isoamylalcohol originates frOm leucine. Isoleucine is dextro- rotatory.
Lysine, CGH14N202. — The three bodies, lysine, arginine and his- tidine, are frequently classed together as the hexone bases. Lysine was the first of the bases discovered. It is a-e-diamino-caproic acid and hence possesses the following structure :
NH2H H H NH2
1 X 1 I I
H-C - C-C-C-C-COOH. H H H H H
It is dextro-rotatory and is found in relatively large amount in casein and gelatin. Lysine is obtained from nearly all proteins but is absent from the vegetable proteins which are soluble in strong alcohol. It is the mother-substance of cadaverin and has never been obtained in crystalline form. Lysine is usually obtained as
1 These balls of so-called impure leucine do contain considerable leucine, but inasmuch as they may contain many other things it is a bad practice to allude to them as leucine.
78
PHYSIOLOGICAL CHEMISTRY.
the picrate which is sparingly soluble in water and crystallizes readily. These crystals are shown in Fig. 27.
Fig. 27.
Lysine Picrate.
Aspartic Acid, C4H7N04. — Aspartic acid is amino-succinic acid and has the following structural formula :
Aspartic Acid.
PROTEINS.
79
The amide of aspartic acid, asparaginic, is very widely distributed in the vegetable kingdom. The crystalline form of aspartic acid is exhibited in Fig. 28.
Aspartic acid has been found among the decomposition products of all the proteins examined, except the protamines. It has not been obtained, however, in very large proportion from any of them. The aspartic acid obtained from protein is laevo-rotatory.
Glutamic Acid, C5H9N04. — This acid is a-amino-normal-glntaric acid and as such bears the following graphic formula:
NH2
CHCOOH
I CH2
CH2COOH.
Glutamic acid is yielded by all the proteins thus far examined, except the protamines, and by most of these in larger amount than any other of their decomposition products. It is yielded in espe- cially large proportion by most of the proteins of seeds, 41.32 per
Fig. 29.
m e
Glutamic Acid.
Reproduced from a micro-photograph made by Prof. E. T. Reichert, of the University
of Pennsylvania.
cent having been obtained very recently by Kleinschmitt from the hydrolysis of hordcin the prolamin of barley. This is the largest
8o
PHYSIOLOGICAL CHEMISTRY.
amount of any single decomposition product yet obtained from any protein except the protamines.1
Glutamic acid and aspartic acid are the only dibasic acids which have thus far been obtained as decomposition products of proteins. As there is an apparent relation between the proportion of these acids and that of ammonia which the different proteins yield it is possible that one of the carboxyl groups of these acids is united with NH2 as an amide, the other carboxyl group being united in poly- peptide union (see page 66) with some other amino acid. This might be represented by the following formula :
R-CHNH-COOH
C0-CHNH9-CH9-CH9-C0NH9.
It has not been definitely proven, however, that this form of link- ing actually occurs.
The glutamic acid, yielded by proteins upon hydrolysis, is dextro- rotatory. Crystals of glutamic acid are reproduced in Fig. 29, page 79.
Proline, C5H9N02. — Proline is a-pyrrolidine-carboxylic acid and possesses the following graphic structure :
HoC CHo
H2C\/CHCOOH. NH
Fig. 30.
L.ffiVO-a-PROLINE.
1Up to this time the yield of 37.33 per cent obtained by Osborne and Harris from gliadin of wheat was the maximum yield
PROTEINS. 8 1
Proline was first obtained as a decomposition product of casein. Proline obtained from proteins is lsevo-rotatory and is the only protein decomposition product which is readily soluble in alcohol. It is also one of the few heterocyclic compounds obtained from pro- teins. Proline was quite recently discovered but has since been found among the decomposition products of all proteins except the protamines. The maximum yield reported is 13.73 Per cent obtained by Osborne and Clapp from the hydrolysis of hordein. The crystal- line form of lav o-a- proline is shown in Fig-. 30, and the copper
Fig. 31.
Copper Salt of Proline.
Reproduced from a micro-photograph made by Prof. E. T. Reichert, of the University
of Pennsylvania.
salt of proline is represented by a micro-photograph in Fig. 31, above. The crystals of the copper salt have a deep blue color but when they lose their water of crystallization they assume a char- acteristic violet color.
Oxyproline, C5H9N03. — Oxyproline was recently discovered by Fischer. It has as yet been obtained from only a few proteins, but this may be due to the fact that only a few have been examined for its presence. Its structure has not yet been established.
Diaminotrihydroxydodecanoic Acid, C12PI2gNo05. — This amino acid was discovered by Fischer and Abderhalden as a product of the hydrolysis of casein. It has thus far been obtained from no other source. It is lsevo-rotatory and its constitution has not been determined.
82 physiological chemistry.
Experiments.
While the ordinary courses in physiological chemistry preclude any extended study of the decomposition products of proteins, the manipulation of a simple decomposition and the subsequent isola- tion and study of a few of the products most easily and quickly obtained will not be without interest.1 To this end the student may use the following decomposition procedure : Treat the protein in a large flask with water containing 3-5 per cent of H2S04 and place it on a water-bath until the protein material has been decomposed and there remains a fine, fluffy, insoluble residue. Filter off this residue and neutralize the filtrate with Ba(OH)2 and BaC03. Filter off the precipitate of BaS04 which forms and when certain that the fluid is neutral or faintly acid,2 concentrate (first on a wire gauze and later on a water-bath) to a syrup. This syrup contains the end-products of the decomposition of the protein, among which are proteoses, peptones, tyrosine, leucine, etc. Add 95 per cent alcohol slowly to the warm syrup until no more precipitate forms, stirring continuously with a glass rod. This precipitate consists of proteoses and peptones. Gather the sticky precipitate on the rod or the sides of the dish and, after warming the solution gently for a few mo- ments, filter it through a filter paper which has not been previously moistened. After dissolving the precipitate of proteoses and pep- tones in water3 the solution may be treated according to the method of separation given on page 114.
The leucine and tyrosine, etc., are in solution in the warm alcoholic
filtrate. Concentrate this filtrate on the water-bath to a thin syrup,
transfer it to a beaker, and allow it to stand over night in a cool
place for crystallization. The tyrosine first crystallizes (Fig. 23,
page 72), followed later by the formation of characteristic crystals
of impure leucine (see Fig. 105, Chapter XX). After examining
these crystals under the microscope, strain off the crystalline material
through fine muslin, heat it gently in a little water to dissolve the leu-
1The procedure here set forth has nothing in common with the procedure by means of which the long line of decomposition products just enumerated are obtained. This latter process is an exceedingly complicated one which is entirely outside the province of any course in physiological chemistry.
2 If the solution is alkaline in reaction at this point, the amino acids will be broken down and ammonia will be evolved.
3 At this point the aqueous solution of the proteoses and peptones may be filtered to remove any BaS04 which may still remain. Tyrosine crystals will also be found here, since it is less soluble than the leucine and may adhere to the proteose-peptone precipitate. Add the crystals of tyrosine to the warm al- cohol filtrate.
PROTEINS. 83
cine (the tyrosine will be practically insoluble) and filter. Concen- trate the filtrate and allow it to stand in a cool place over night for the crude leucine to crystallize. Filter off the crystals and use them in the tests for leucine given on page 84. The crystals of tyrosine remaining on the paper from the first filtration may be used in the tests for tyrosine as given below. If desired, the tyrosine and leucine may be purified by recrystallizing in the usual manner. Habermann has suggested a method of separating leucine and tyrosine by means of glacial acetic acid.
Experiments on Tyrosine.
Make the following tests with the tyrosine crystals already pre- pared or upon some pure tyrosine furnished by the instructor.
1. Microscopical Examination. — Place a minute crystal of ty- rosine on a slide, add a drop of water, cover with a cover glass, and examine microscopically. Now run more water under the cover glass and warm in a bunsen flame until the tyrosine has dissolved. Allow the solution to cool slowly then examine again microscopically and compare the crystals with those shown in Fig. 23, page 72.
2. Solubility. — Try the solubility of very small amounts of tyro- sine in cold and hot water, cold and hot 95 per cent alcohol, dilute NH4OH, dilute KOH and dilute HC1.
3. Sublimation. — Place a little tyrosine in a dry test-tube, heat gently and notice that the material does not sublime. How does this compare with the result of Experiment 3 under Leucine?
4. Hoffman's Reaction. — This is the name given to Millon's reaction when employed to detect tyrosine. Add about 3 c.c. of water and a few drops of Millon's reagent to a little tyrosine in a test-tube. Upon dissolving the tyrosine by heat the solution gradu- ally darkens and may assume a dark red color. What group does this test show to be present in tyrosine?
5. Piria's Test. — Warm a little tyrosine on a watch glass on a boiling water-bath for 20 minutes with 3-5 drops of cone. H2S04. Tyrosine sulphuric acid is formed in the process. Cool the solution and wash it into a small beaker with water. Now add CaC03 in substance slowly with stirring, until the reaction of the solution is no longer acid. Filter, concentrate the filtrate and add to it a few drops (avoid an excess) of very dilute neutral ferric chloride. A purple or violet color, due to the formation of the ferric salt of tyrosine-sulphuric acid, is produced. This is one of the most satis- factory tests for the identification of tyrosine.
84 PHYSIOLOGICAL CHEMISTRY.
6. Morner's Test. — Add about 3 c.c. of Morner's reagent1 to a little tyrosine in a test-tube, and gently raise the temperature to the boiling-point. A green color results.
Experiments on Leucine.
Make the following tests upon the leucine crystals already pre- pared or upon some pure leucine furnished by the instructor.
1, 2 and 3. Repeat these experiments according to the directions given under Tyrosine (page 83).
1 Morner's reagent is prepared by thoroughly mixing i volume of formalin. 45 volumes of distilled water and 55 volumes of concentrated sulphuric acid.
CHAPTER V.
PROTEINS: THEIR CLASSIFICATION AND PROPERTIES.
From what has already been said in Chapter IV, regarding the protein substances it will be recognized that the grouping of the diverse forms of this class of substances in a logical manner is not an easy task. The fats and carbohydrates may be classified upon the fundamental principles of their stereo-chemical relationships whereas such a system of classification in the case of the proteins is absolutely impossible since, as we have already stated, the mole- cular structure of these complex substances is unknown. Because of the diversity of standpoint from which the proteins may be viewed, relative to their grouping in the form of a logically classified series, it is obvious that there is an opportunity for the presen- tation of classifications of a widely divergent character. The fact that there were until recently at least a dozen different classifica- tions which were recognized by various groups of English-speaking investigators, emphasizes the difficulties in the way of the individual or individuals who would offer a classification which should merit universal adoption. Realizing the great handicap and disadvantage which the great diversity of the protein classifications was forcing upon the workers in this field the British Medical Association re- cently drafted a classification which appealed to that body of scien- tists as fulfilling all requirements and presented it for the consid- eration of the American Physiological Society and the American Society of Biological Chemists. The outcome of this has been that there are now only two protein classifications which are recognized by English-speaking scientists, one the British Classification the other the American Classification. These classifications are very similar and doubtless will ultimately be merged into a single classification. In our consideration of the proteins we shall conform in all de- tails to the American Classification. In this connection we will say, however, that we feel that the British Medical Association has strong grounds for preferring the use of the term scleroprotcins for albuminoids and chromoproteins for haemoglobins. The two classi- fications are as follows :
85
86 PHYSIOLOGICAL CHEMISTRY.
CLASSIFICATION OF PROTEINS ADOPTED BY THE AMERICAN PHYSIOLOGICAL SOCIETY AND THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS.
I. SIMPLE PROTEINS.
Protein substances which yield only a-amino acids or their de- rivatives on hydrolysis.
(a) Albumins. — Soluble in pure water and coagulable by heat, e. g., ovalbumin, serum albumin, lactalbumin, vegetable albumins.
(b) Globulins. — Insoluble in pure water but soluble in neutral solutions of salts of strong bases with strong acids,1 e. g., serum globulin, ovo globulin, edestin, amandin and other vegetable globu- lins.
(c) Glutelins. — Simple proteins insoluble in all neutral solvents but readily soluble in very dilute acids and alkalis,2 e. g., glutenin.
(d) Alcohol-soluble proteins (Prolamins).3 — Simple proteins soluble in 70-80 per cent alcohol, insoluble in water, absolute alcohol and other neutral solvents,4 e. g., zein, gliadin, hordein and bynin.
(e) Albuminoids. — Simple proteins possessing a similar struc- ture to those already mentioned, but characterized by a pronounced insolubility in all neutral solvents,5 e. g., elastin, collagen, keratin.
(/) Histones. — Soluble in water and insoluble in very dilute ammonia, and, in the absence of ammonium salts, insoluble even in excess of ammonia; yield precipitates with solutions of other pro- teins and a coagulum on heating which is easily soluble in very dilute acids. On hydrolysis they yield a large number of amino acids among which the basic ones predominate. In short histones are basic proteins which stand between protamines and true pro- teins, e. g., globin, thymus histone, scombrone.
1 The precipitation limits with ammonium sulphate should not be made a basis for distinguishing the albumins from the globulins.
2 Such substances occur in abundance in the seeds of cereals and doubtless represent a well-defined natural group of simple proteins.
3 The name prolamins has been suggested for these alcohol-soluble proteins by Dr. Thomas B. Osborne (Science, 1908, XXVIII, p. 417). It is a very- fitting term inasmuch as upon hydrolysis they yield particularly large amounts of proline and ammonia.
4 The subclasses defined (a, b, c, d,) are exemplified by proteins obtained from both plants and animals. The use of appropriate prefixes will suffice to indicate the origin of the compounds, e. g., ovoglobulin, lactalbumin, etc.
5 These form the principal organic constituents of the skeletal structure of animals and also their external covering and its appendages. This definition does not provide for gelatin which is, however, an artificial derivative of collagen.
PROTEINS. 87
{g) Protamines. — Simpler polypeptides than the proteins in- cluded in the preceding groups. They are soluble in water, uncoag- ulable by heat, have the property of precipitating aqueous solutions of other proteins, possess strong basic properties and form stable salts with strong mineral acids. They yield comparatively few amino acids, among which the basic ones predominate. They are the simplest natural proteins, e. g., sahnine, sturine, clupeine, scorn- brine.
II. CONJUGATED PROTEINS.
Substances which contain the protein molecule united to some other molecule or molecules otherwise than as a salt.
(a) Nucleoproteins. — Compounds of one or more protein mole- cules with nucleic acid, e. g., cytoglobulin, nucle ohist one.
(b) Glycoproteins. — Compounds of the protein molecule with a substance or substances containing a carbohydrate group other than a nucleic acid, e. g., mucins and mucoids {osseomucoid, tendomu- coid, ichthulin, helicoprotein) .
(c) Phosphoproteins. — Compounds of the protein molecule with some, as yet undefined, phosphorus-containing substances other than a nucleic acid or lecithin,1 e. g., caseinogen, vitellin.
{d) Haemoglobins. — Compounds of the protein molecule with hsematin, or some similar substance, e. g., hcemoglobin, hcemocya- nin.
{e) Lecithoproteins. — Compounds of the protein molecule with lecithins, e. g., lecithans, phosphatides.
III. DERIVED PROTEINS.
I. Primary Protein Derivatives. Derivatives of the protein molecule apparently formed through hydrolytic changes which involve only slight alteration of the pro- tein molecule.
(a) Proteans. — Insoluble products which apparently result from the incipient action of water, very dilute acids or enzymes, e. g., myosan, edestan.
(b) Metaproteins. — Products of the further action of acids and alkalis whereby the molecule is so far altered as to form products soluble in very weak acids and alkalis but insoluble in neutral fluids, e. g., acid metaprotein {acid albuminate) , alkali mctaprotein {alkali albuminate) .
1 The accumulated chemical evidence distinctly points to the propriety of classi- fying the phosphoproteins as conjugated compounds, i. c, they are possibly esters of some phosphoric acid or acids and protein.
55 PHYSIOLOGICAL CHEMISTRY.
(c) Coagulated Proteins. — Insoluble products which result from (i) the action of heat on their solutions, or (2) the action of alcohol on the protein.
2. Secondary Protein Derivatives.1
Products of the further hydrolytic cleavage of the protein molecule.
(a) Proteoses. — Soluble in water, non-coagulable by heat, and precipitated by saturating their solutions with ammonium — or zinc sulphate,2 e. g., protoproteose, deuteroproteose.
(b) Peptones. — Soluble in water, non-coagulable by heat, but not precipitated by saturating their solutions with ammonium sul- phate,3 e. g., antipeptone, ampho peptone.
(c) Peptides. — Definitely characterized combinations of two or more amino acids, the carboxyl group of one being united with the amino group of the other with the elimination of a molecule of water,4 e. g., dipeptides, tripeptides, tetrapeptides, pentapeptides.
CLASSIFICATION OF PROTEINS ADOPTED BY THE BRITISH MEDICAL ASSOCIATION.
I. Simple Proteins.
1. Protamines, e. g., salmine, clupeine.
2. Histones, e. g., globin, scombrone.
3. Albumins, e. g., ovalbumin, serum albumin, vegetable albu- mins.
4. Globulins, e. g., serum globulin, ovoglobulin, vegetable glob- ulins.
5. Glutelins, e. g., glutenin.
6. Alcohol-soluble proteins, e. g., zein, gliadin.
7. Scleroproteins, e. g., elastin, keratin.
8. Phosphoproteins, e. g., caseinogen, vitellin.
1 The term secondary hydrolytic derivatives is used because the formation of the primary derivatives usually precedes the formation of these secondary derivatives.
2 As thus defined, this term does not strictly cover all the protein derivatives commonly called proteoses, e. g., heteroproteose and dysproteose.
8 In this group the kyrines may be included. For the present it is believed that it will be helpful to retain this term as defined, reserving the expression peptide for the simpler compounds of definite structure, such as dipeptides, etc.
4 The peptones are undoubtedly peptides or mixtures of peptides, the latter term being at present used to designate those of definite structure.
PROTEINS. 89
II. Conjugated Proteins.
1. Oncoproteins, e. g., mucins, mucoids.
2. Nucleoproteins, e. g., nucleohistone, cytoglobulin.
3. Chromoproteins, e. g., hemoglobin, hccmocyanin.
III. Products of Protein Hydrolysis.
1. Infraproteins, e. g., acid infraprotein (acid albuminate) , alkali infraprotein (alkali albuminate).
2. Proteoses, e. g., protoproteose, heteroproteose, deuteroproteose.
3. Peptones, e. g., ainphopeptone, antipeptone.
4. Polypeptides, e. g., dipeptides, tripeptides, tetrapeptides.
CONSIDERATIONS OF THE VARIOUS CLASSES OF PROTEINS.
SIMPLE PROTEINS.
The simple proteins are true protein substances which, upon hy- drolysis, yield only a-amino acids or their derivatives. "Although no means are at present available whereby the chemical individual- ity of any protein can be established, a number of simple proteins have been isolated from animal and vegetable tissues which have been so well characterized by constancy of ultimate composition and uniformity of physical properties that they may be treated as chemical individuals until further knowledge makes it possible to characterize them more definitely." Under simple proteins we may class, albumins, globulins, glutelins, prolamins, albuminoids, his- tones and protamines.
ALBUMINS.
Albumins constitute the first class of simple proteins and may be defined as simple proteins which are coagulable by heat and soluble in pure (salt-free) water. Those of animal origin are not precipitated upon saturating their neutral solutions at 300 C. with sodium chloride or magnesium sulphate, but if a saturated solution of this character be acidified with acetic acid the albumin precipi- tates. All albumins of animal origin may be precipitated by sat- urating their solutions with ammonium sulphate.1 They may be
1 In this connection, Osborne's observation that there are certain vegetable albumins which are precipitated by saturating their solutions with sodium chlor- ide or magnesium sulphate or by half-saturating with ammonium sulphate, is of interest.
90 PHYSIOLOGICAL CHEMISTRY.
thrown out of solution by the addition of a sufficient quantity of a mineral acid, whereas a weak acidity produces a slight precipitate which dissolves upon agitating the solution. Metallic salts also possess the property of precipitating albumins, some of the precipi- tates being soluble in excess of the reagent whereas others are in- soluble in such an excess. Of those proteins which occur native the albumins contain the highest percentage of sulphur, ranging from 1.6 to 2.5 per cent. Some albumins have been obtained in crystalline form, notably egg albumin, serum albumin and lactal- bumin but the fact that they may be obtained in crystalline form does not necessarily prove them to be chemical individuals.
GENERAL COLOR REACTIONS OF PROTEINS.
These color reactions are due to a reaction between some one or more of the constituent radicals or groups of the complex protein molecule and the chemical reagent or reagents used in any given test. Not all proteins contain the same groups and for this reason the various color tests will yield reactions varying in intensity of color according to the nature of the groups contained in the par- ticular protein under examination. Various substances not pro- teins respond to certain of these color reactions and it is therefore essential to submit the material under examination to several tests before concluding definitely regarding its nature.
TECHNIQUE OF THE COLOR REACTIONS.
i. Millon's Reaction. — To 5 c.c. of a dilute solution of egg albumin in a test-tube add a few drops of Millon's reagent. A white precipitate forms which turns red when heated. This test is a particularly satisfactory one for use on solid proteins, in which case the reagent is added directly to the solid substance and heat applied, which causes the substance to assume a red color. Such proteins as are not precipitated by mineral acids, for example certain of the proteoses and peptones, yield a red solution instead of a red precipitate.
The reaction is due to the presence of the hydroxy-phenyl group, — C6H4OH, in the protein molecule and certain non-proteins such as tyrosine, phenol (carbolic acid) and thymol also respond to the reaction. Inasmuch as the tyrosine grouping is the only hy- droxy-phenyl grouping which has definitely been proven to be
PROTEINS. 91
present in the protein molecule it is evident that protein substances respond to Millon's reaction because of the presence of this tyro- sine complex. The test is not a very satisfactory one for use in solutions containing inorganic salts in large amount, since the mer- cury of the Millon's reagent1 is thus precipitated and the reagent rendered inert. This reagent is therefore never used for the detec- tion of protein material in the urine.
2. Xanthoproteic Reaction. — To 2-3 c.c. of egg albumin solu- tion in a test-tube add concentrated nitric acid. A white precipi- tate forms, which upon heating turns yellow and finally dissolves, imparting to the solution a yellow color. Cool the solution and carefully add ammonium hydroxide, potassium hydroxide or sod- ium hydroxide in excess. Note that the yellow color deepens into an orange. This reaction is due to the presence in the protein molecule of the phenyl group, with which the nitric acid forms certain nitro modifications. The particular complexes of the pro- tein molecule which are of especial importance in this connection are those of tyrosine, phenylalanine and tryptophane. The test is not a satisfactory one for use in urinary examination because of the color of the end-reaction.
3. Adamkiewicz Reaction. — Thoroughly mix 1 volume of con- centrated sulphuric acid and 2 volumes of acetic acid in a test-tube, add a few drops of egg albumin solution and heat gently. A reddish-violet color is produced. Gelatin does not respond to this test. This reaction shows the presence of the tryptophane group (see next experiment). The test depends upon the presence of glyoxylic acid, CHO ■ COOH + H20 or CH(OH)2COOH, in the reagents. This is shown by the failure to secure a positive reaction when acetic acid free from glyoxylic acid is used.
Rosenheim has recently advanced the view that the reaction may be due to the presence of oxidizing agents such as nitrous acid and ferric salts in the sulphuric acid.
4. Hopkins-Cole Reaction. — Place 1-2 c.c. of egg albumin solu- tion and 3 c.c. of glyoxylic acid, CHO " COOH -j- H20 or CH(OH)2COOH, solution (Hopkins-Cole reagent2) in a test-tube
1 Millon's reagent consists of mercury dissolved in nitric acid containing some nitrous acid. It is prepared by digesting one part (by weight) of mercury with two parts (by weight) of HN03 (sp. gr. 1.42) and diluting the resulting solution with two volumes of