GIFT OF t UNIVERSITY OF CALIFORNIA. FINAL EXAMINATION OF VICTOR BIRCKNER M.S., (UNIVERSITY OF CALIFORNIA) 1911 FOR THE DEGREE OF DOCTOR OF PHILOSOPHY, MONDAY, APRIL 29, 1912, AT 2:00 P.M., IN THE RUDOLPH SPRECKELS PHYSIOLOGICAL LABORATORY. SUB-COMMITTEE IN CHARGE: PROFESSOR T. BRAILSFORD ROBERTSON. PROFESSOR W. A. SETCHELL. PROFESSOR A. R. MOORE. PROFESSOR S. S. MAXWELL. PROFESSOR w. L. JEPSON. ( Physiological Chemistry. MAJOR SUBJECT < ( Research Work on Sugar Fermentations. I. Advanced Chemical Biology. Professor Robertson. II. Research Work in Physiological Chemistry. Professor Robertson. MINOR SUBJECT: BOTANY I. A general study of the orders of the spore-bearing green plants from the points of view of structure, development, and economic importance. Professor Setchell. II. Experimental Plant Physiology. Research work on some factors influencing the germina- tion of seeds. Professor Moore. OTHER SUBJECTS I. Research in Chemico-Agricultural Technology. Professor Shaw. II. Lectures on Dairy Chemistry. Professor Jaffa and Mr. McCharles. III. Advanced work in the Chemical and Microscopical investigation of Fertilizers. Professor Burd. IV. Water Supply for Irrigation. Conservation and Use of Water. Professor Chandler. THESIS : On the Oxydations and Cleavages of Glucose. Yeast Glucase, a new Glucolytic Ferment. SUMMARY (1) A systematic review has been given in the first part of the paper of some recent advances of our knowledge of glucose oxydations and cleavages, both outside and inside of the organism. (2) In the second part of this article a ferment has been described which occurs in the California steam beer yeast under certain conditions, and which has the property of accelerating the decomposition of glucose at an elevated temperature. (3) This new ferment is not identical with zymase. It acts preferably at. a temperature <>!' 70 C. It causes no gas formation, and yields no alcohol. (4) Its action on glucose manifests itself by a rapid darkening of the mixture, a strongly acid reaction, a gradual formation of a carbon-like, solid settlement, and the development of an odor similar to caramel. (5) The ferment may be extracted from a yeast powder (Dauerhewe) which is best obtained by treating the cells with aethyl-alcohol. (6) From the watery extract the yeast glucase may be obtained and purified by repeated precipitation with alcohol; but this process always involves a weakening of the ferment. (7) Yeast glucase is -very stable in aqueous solution if kept at room temperature under sterile conditions. Boiling for one minute does not destroy its activity. (8) Yeast glucase shows activity in neutral or acid solution against glucose, polyphenols, and lactates. The preparation does not contain tyrosinase, nor does it act as a peroxydase against glucose. (9) The ferment preparation gives a strong pyrrol reaction (Neuberg). (10) Yeast glucase shows some relationship to the oxydases, but with regard to its main function, it is to be classed together with zymase in a group which stands separately from the oxydases and the hydrolytic ferments, and to which uler has applied the names "G-arungs fermente." (11) The transformation products of glucose, resulting from the action of this ferment, are largely acids, none of which has so far been clearly identified. However, among the cleavage products of the sugar, the presence of pentose and formaldehyde was ascertained, which is of interest with regard to the recent work of W. Lob. UNIVERSITY OF CALIFORNIA PUBLICATIONS ' V l| *, ; PHYSIOLOGY Vol. 4, No. 16, pp. 115-183 September 20, 1912 ON THE OXYDATIONS AND CLEAVAGES OF GLUCOSE. YEAST GLUCASE, A NEW GLUCOLYTIC FERMENT BY VICTOR BIRCKNER UNIVERSITY OF CALIFORNIA PRESS BERKELEY UNIVERSITY OF CALIFORNIA PUBLICATIONS Note. The University of California Publications are offered in exchange for the publi- cations of learned societies and institutions, universities and libraries. Complete lists of all the publications of the University will be sent upon request. For sample copies, lists of publications and other information, address the Manager of the University Press, Berkeley, California, U. S. A. All matter sent in exchange should be addressed to The Exchange Department, University Library, Berkeley, California, U. S. A. OTTO HAREASSOWITZ, R. FRIEDLAENDER & SOHN, LEIPZIG. BERLIN. Agent for the series in American Arcn- Agent for the series in American Arch- aeology and Ethnology, Classical Philology, aeology and Ethnology, Botany, Geology, Education, Modern Philology, Philosophy, Mathematics, Pathology, Physiology, Zool- Psychology. ogy, and Memoirs. PHYSIOLOGY S. S. Maxwell, Editor. Price per volume $2. Cited as Univ. Calif. Publ. Physiol. VoLl. 1. On a Method by which the Eggs of a Sea-urchin (Strngylocentrotu purpuratus) can be Fertilized with the Sperm of a Starfish (Atteriat ochracea), by Jacques Loeb. Pp. 1-3. April, 1903._ 05 2. On the Mechanism of the Action of Saline Purgatives, and the Counteraction of their Effect by Calcium, by John Bruce Mac- Callum. Pp. 6-6. May, 1903 05 S. Artificial Parthenogenesis in Molluscs, by Jacques Loeb. Pp. 7-9. August, 1903 - _ .05 4. The Relations of Biology and the Neighboring Sciences, by Wilhelm Ostwald. Pp. 11-31. October, 1903 25 5. The Limitations of Biological Research, by Jacques Loeb. Pp. 33-37. October, 1903 __ 05 6. The Fertilization of the Egg of the Sea-urchin by the Sperm of the Starfish, by Jacques Loeb. Pp. 39-53. November, 1903 15 7. On the Relative Toxicity of Distilled Water, Sugar Solutions and Solutions of the various Constituents of the Sea-water for Marine Animals, by Jacques Loeb. Pp. 55-69. November, 1903. 8. On the Segmental Character of the Respiratory Center in the Medulla Oblongata of Mammals, by Jacques Loeb. Pp. 71-75. November, 1903. Nos. 7 and 8 in one cover .25 9. On the Production and Suppression of Glycosuria in Rabbits through Electrolytes (a preliminary communication), by Martin H. Fischer. Pp. 77-79. December, 190SL- .'. ~ _.. .05 10. On the Influence of Calcium and Barium on the Flow of Urine (a preliminary communication), by John Bruce MacCallum. Pp. 81-82. January, 1904 .05 11. Further Experiments on the Fertilization of the Egg of the Sea-urchin with Sperm of various species of Starfish and a Hclothurian, by Jacques Loeb. Pp. 83-85. February, 1904 .05 12. On the Production aud Suppression of Glycosuria in Rabbits through Electro.\Ftos (sepoijd, , communication), by Martin H. Fischer. Pp. 87-'i;i.. V Febr>o&rjr,: rt;3G4.; 30 13. The Influence of Saline "Purgatives on Loops of Intestine Removed from, tfcp 'B$ifcr ? bjr'J-ohik Bruce MacCallum. Pp. 115-123. March, 1903-.'. :;'','; ; ; ; . '. , 14. The Secretion of Sugar into the Intestine Caused by Intravenous Saline Infusions, by John Bruce MacCallum. Pp. 125-137. March, 1904. Nos. IS and 14 in one cover 20 15. On the Influence of the Reaction of the Sea-water on the Regeneration and Growth of Tubularians, by Jacques Loeb. Pp. 139-147. April, 1904 _ .10 16. The Possible Influence of the Amphoteric Reaction of Certain Colloids upon the Sign of their Electrical Charge in the Presence of Acid and Alkalis, by Jacques Loeb. Pp. 149-150. May, 1904. 17. Concerning Dynamic Conditions which contribute toward the Deter- mination of the Morphological Polarity of Organisms (first com- munication), by Jacques Loeb. Pp. 151-161. 7 text figures. May, 1904. Nos. 16 and 17 in one cover _ 16 18. The Action of Cascara Sagrada (a preliminary communication), by John Bruce MacCallum. Pp. 163-164. May, 1904 05 19. Artificial Parthenogenesis and Regular Segmentation in an Annelid (Ophelia), by G. Bullot. 13 text figures. Pp. 165-174. June, 1S04-. .10 20. On the Action of Saline Purgatives in Rabbits and the Counteraction of their Effect by Calcium (second communication), by John Bruce MacCaUum. Pp. 175-185. July, 1904, UNIVERSITY OF CALIFORNIA PUBLICATIONS IN PHYSIOLOGY Vol. 4, No. 16, pp. 115-183 September 20, 1912 ON THE OXYDATIONS AND CLEAVAGES OF GLUCOSE. YEAST GLUCASE, A NEW GLUCOLYTIC FERMENT* BY VICTOR BIECKNEB CONTENTS PAGE Introduction H6 Part I. Modern Viewpoints on the Mechanism of the Oxydations and cleavages of Glucose Inside and Outside of the Living Organism 119 A. Destruction of glucose by chemical or physical agencies of known character 119 1. The structure of the glucose molecule 119 2. The amphoteric character of the sugars 122 3. The action of radiant energy on glucose 126 (a) The electrolysis of sugars Lob's theory 126 (5) The action of light 136 4. The action of alkalies and acids on glucose 138 B. The oxydations and cleavages of glucose through the action of more or less unknown agencies 144 Part II. Yeast Glucase, a New Glucolytic Ferment 158 1. The ferment as first observed and recognized 158 2. Experiments with hydroquinone 165 3. Method of preparing the yeast powder 171 4. Method of extracting the ferment 172 5. Attempts of further purification 172 6. Properties of the yeast extract 174 7. Studies on the products of glucose fermentation 176 Conclusions . 182 * Presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of California. 244546 . '** "*,, ** * 1 : s/:V :!.,:>,-.. 116 University of California Publications in Physiology [VOL. 4 INTRODUCTION The work of Emil Fischer 1 on the artificial synthesis of sugars in 1890, marks the beginning of a new period of bio- chemical research. The foundations of an accurate study of the carbohydrates were laid. This study was taken up immediately by a large number of workers, and numerous new facts were brought to light during the last twenty years, partly through Fischer's own efforts. It is therefore not surprising that our conceptions to-day are already slightly beyond the original conceptions of the great investigator. The carbohydrates are considered as being among the most important food materials of the animal body. They form a rather uniform class of substances which, especially in its lower members, is well understood as far as chemical composi- tion and properties are concerned. We are confronted by the greatest difficulties, however, when trying to investigate in what way these substances are naturally built up and broken down in the living organism. The central figure in the whole carbohydrate metabolism of any living being is the rather simple looking substance glucose, of the empirical formula C 6 H 12 O G . This substance is formed in enormous quantities in the green parts of the plant by a rapid reduction of the carbon dioxide of the air, and subsequent polymerization processes. Through further condensations of glucose molecules, the plant synthesizes and stores up at the proper places those immense masses of reserve materials, to which, either directly or indirectly, the whole animal world owes its subsistence. On its way through the animal body, the complex carbo- hydrate molecule undergoes changes similar to those by which its synthesis was brought about in the plant, only in the opposite direction. The molecule is first broken down through the action of certain ferments of the alimentary canal to the state of 1 Emil Fischer, several articles in Ber. DeutscJi. chem. Ges., vol. 23 and following; collected in Untersuchungen iiber Kohlcnhydrate und Fermcnte (Berlin, J. Springer, 1884-1908). 1912] Bircknf.r: Glucose Oxydations 117 glucose, whereupon resorption by the blood stream can take place. After thus being taken into the circulating system, the glucose molecule undergoes various changes, the nature of which is very little understood. Part of the sugar we find again in a polmerized form as glycogen in the liver and in the muscles. Its main portion, however, serves as substrate for that slow oxydation (combustion) process which is constantly going on in every cell of the body, more extensively in the lungs, and the final products of which we know to be carbon dioxyde and water. The synthetic part of this carbon cycle (CO 2 > glucose -- polysaccharide) is almost exclusively a function of the green plant, while the reverse reaction (polysaccharide glucose COo) is constantly going on in both plants and animals. The ease with which all these changes are apparently brought about in the living cells and tissues, at a rather low temperature and in a practically neutral medium, together with the fact that we- have a perfectly clear understanding of the physical and chemical properties of the chief substance involved, and that we can prepare this substance free from impurities, have been a constant stimulus for the investigator for a large number of years. Numerous attempts have been made to gain an insight into the ways and means by which nature brings about these funda- mental transformations, a knowledge of which would mean an important step towards the final solution of the problem of artificial synthesis of organic matter in more economic ways. It is of equal importance for this purpose whether we study the synthetic process as such, or whether we try to follow the glucose molecule as it is being broken down in the organism. The latter way of proceeding is perhaps of more immediate interest in view of its bearing on the physiology and pathology of our own body. Progress is being made, however, in both directions, and, although at present our knowledge on either side does not go far beyond the stage of a hypothesis, it is to be noticed after the advances of recent years that the workers at both sides of the problem are approaching each other more and more closely, and that the unexplored area between them is being constantly diminished. 118 University of California Publications in Physiology [VOL. 4 I shall try in the first part of this paper to review briefly the more recent attempts that have been made with the object of approaching the problem of " glucolysis. " By this term, which is frequently to be met with in the newer literature, may be understood 2 all exothermic cleavages of the glucose molecule which furnish energy to the body, no matter if through the interaction of oxygen, or in its absence. In some English articles 3 I notice the word being spelled "glycolysis" in analogy to the German form. As this term, however, is apparently already in use for a different process, 4 I prefer to write the word ' ' glucolysis. ' ' 2 According to W. Lob, Beitrage zur Frage der Glycolyse I, Biochem. Zeitschrift, vol. 29, p. 317, 1910. 3 E.g., L. Henderson, The instability of glucose at the temperature and alkalinity of the body, Journ. Biol. Chem., vol. 10, p. 4, 1911. 4 Gould, Medical Dictionary, p. 524, 194. 1912 ] Birckner: Glucose Oxydations 119 PART I MODERN VIEWPOINTS ON THE MECHANISM OF THE OXYDATIONS AND CLEAVAGES OF GLUCOSE INSIDE AND OUTSIDE OF THE LIVING ORGANISM The matter which is being taken up on the following pages represents the first trial, to my knowledge, to bring together and arrange systematically all important experimental results of recent years which have helped to elucidate somewhat the phenomena involved in the decomposition of grape sugar. The subject which I shall try to cover has, undoubtedly, not yet arrived at the stage where the available data have the full value of facts, and it would hardly pay at present to enter into a lengthy discussion of all experimental results obtained and of every hypothesis advanced. Still, as stated, the achievements of recent years are rather encouraging, and as the problem is of equal importance for several branches of science, the literature on the subject' is becoming extensive, the problem being fre- quently treated from very different points of view. For a person interested anew in this study it is already difficult to look over the entire field; and being aware of this circumstance, I dare to hope that the following sketch of the present stand of the question will not be useless. A. DESTRUCTION OF GLUCOSE BY CHEMICAL OR PHYSICAL AGENCIES OF KNOWN CHARACTER 1. The Structure of the Glucose Molecule A few words may be said at the outset with regard to the configuration formula of glucose, as a more intimate inquiry into the phenomena of multirotation, together with a better understanding of the properties and the chemical constitution of 120 University of California Publications in Physiology [VOL. 4 the glucosides, have brought to light during recent years many important facts which for various reasons demand a thorough revision of our present views, and to which the biologist should not fail to pay due attention. As the first oxydation product of normal hexane, glucose is usually represented by the formula CH 2 (OH). CH(OH). CH(OH). CH(OH) CH(OH). CHO, this open-chain formula bearing an aldehyde group at one end, and an alkohol radicle at the other. It has, however, long been noticed that the substance is far less active chemically than would be expected from a hydroxy- aldehyde. Tollens, 1 therefore, as early as 1883, suggested to express this relatively high stability by representing the mole- cule through a ring formula, the ring containing four of the six carbon atoms and one atom of oxygen. As a result of the researches of Emil Fischer, 2 Tanret, 3 E. F. Armstrong, 4 and others 5 on the two stereoisomeric forms of glucose and their relations to the corresponding methyl-glucosides, the glucose formula which is now almost generally agreed upoii 5a is the following : 1 B. Tollens, Das Verhalten der Dextrose zu ammoniakalischer Silber- lo'sung, Ber. Deutsch. chem. Ges., vol. 16, p. 921, 1883. 2 E. Fischer, Einige Sauren der Zuckergruppe, Ber. Deutscli. chem. Ges., vol. 23, p. 2625, 1890; tiber die Glucoside der Alkohole, ibid., vol. 26, p. 2400, 1893; ibid., vol. 28, p. 1145, 1895. 3 C. Tanret, Les modifications moleculaires du glucose, Bull. Soc. Chim., (m), vol. 13, pp. 625, 728, 1895; Compt. rend., vol. 120, pp. 1060-1062, 1895; Les modifications moleculaires et la multirotation des sucres, Bull. Soc. Chim., (m), vol. 15, pp. 195, 349, 1896; Les transformations des sucres a multirota- tion, Bull. Soc. Chim. (m), vol. 33, p. 337, 1905. 4 E. F. Armstrong, Studies on Enzyme Action. I. The correlation of the stereoisomeric a- and /3-glucosides wtih the corresponding glucoses, Journ. Chem. Soc., vol. 83, p. 1305, 1903. 5 E.g., L. J. Simon, Sur la constitution du glucose, Compt. rend., vol. 132, pp. 487, 596, 1901. For details and other literature on this paragraph see E. F. Armstrong, The Simple Carbohydrates and the Glucosides. Monographs on Biochemistry (London, Longmans, Green & Co., 1910), p. 3 and follow- ing, and pp. 93-94. 5a See for instance Emil Fischer, Ber. d. Deutsch. chem. Ges., vol. 45, p. 461, 1912. 1912] Birckner: Glucose Oxydations 121 OH CH. CH(OH). CH 2 (OH) It is seen that in this closed-chain formula the molecule has five asymmetric carbon atoms as against four in the empirical formula. It is further seen that the carbon atom at the left end of the pentaphane ring does not carry an aldehyde group any more. As a matter of fact, however, glucose in aqueous solution displays aldehydic properties. To account for this behavior it is to be assumed that in aqueous solution, part of the molecules undergo a sort of intramolecular hydrolysis, by which the pentaphane ring is ruptured and the aldehyde potential of the carbon atom to the left set free. In an aqueous solution of glucose, therefore, according to E. F. Armstrong, 6 the following equilibrium is established : H OH V HC(OH) -f H,O O (OH)CH HC HC(OH) H 2 C(OH) Closed Ring (y-oxide modification) H OH Y HC(OH) OH (OH)CH OH HC/ I HC(OH) H 2 C(OH) Aldehydrol O H V HC(OH) OH HC(OH) H 2 C(OH) Aldehyde (open chain modification) Supposing now the aldehyde be attacked by an oxydizing .agent such as Fehling's solution; its destruction will disturb the equilibrium, causing the heptahydric alcohol (aldehydrol), which is formed intermediately, to change into the aldehyde, and this again causing a fresh formation of aldehydrol from the closed-ring form. In this way, as the reaction proceeds, all of the closed-ring modification will be finally changed into aldehyde. E. F. Armstrong, The Simple Carbohydrates, etc., p. 4. 122 University of California Publications in Physiology [VOL. 4 On the other hand, if a substitution or esterification product (such as a glucoside) is to be formed, the reaction will take place at the closed-ring side of the system, and all the aldehyde will be transformed to the closed-ring modification. The reaction, therefore, is a reversible one. Somewhat similar relations are assumed by Lowry 7 to under- lie the isomeric change of a- into /3-glucose and its reverse. To the fact that in aqueous solution glucose always exists in these two stereo-isomeric modifications, which form an equilibrated mixture and can readily be converted into one another, E. F. Armstrong 8 attributes considerable biological significance. No experimental data are at present available with regard to this point. 2. The Amphoteric Character of the Sugars An aqueous solution of pure glucose, if kept under sterile conditions, shows very high stability. At room temperature it may be kept for long periods before the solution turns slightly yellow, or before a fall of the optical activity or of the copper-reducing power becomes distinctly noticeable. At " a higher temperature these changes occur more rapidly, as the dissociation of the water itself is considerably increased. Still at a temperature of 70 C. a 10 per cent solution of pure glucose does not show the slightest change in appearance and color for at least three weeks (it was the question of coloration which at the beginning of my work interested me more than anything else, as will be seen in the second part of this paper.) Experiments of Kullgren 9 had already demonstrated the strongly depressing effect that cane-sugar had on the velocity of saponification of aethyl acetate by sodium hydroxide, and thereby established the acid character of this sugar in the presence of alkali. E. Cohen 10 on repeating these experiments with different ? T. M. Lowry, Studies of Dynamic Isomersism. The mutarotation of glucose, Journ. Chem. Soc., vol. 75, p. 213, 1899; ibid., vol. 83, p. 1314, 1903. s E. F. Armstrong, The Simple Carbohydrates, etc., p. 20. Svensk. Akad. Handl., vol. 24, 1898 ; cf . E. Cohen, Studien iiber die Inversion, Zeitschr. f. Physilc. Chemie, vol. 37, p. 69, 1901. 10 E. Cohen, loc. cit. 1912] Birckner: Glucose Oxydations 123 sugars found that they all show more or less the same behavior. From his tables (his results had already been published in Dutch a year or two before) Osaka 11 succeeded in calculating the dissociation-constant of glucose. He obtained the value of 5.9 X 10- 13 at 25 C. On the other hand, it could be followed from the work of Baeyer and Villiger, 12 Cohen, 13 Walden, 14 Walker, 15 and recently of Stieglitz 16 that glucose behaves like a very weak base in the presence of acids, uniting with the acid at the aldehydic oxygen atom to form an oxonium salt. This salt, as Bunzel and Mathews 17 point out, dissociates, though only very slightly, into positive glucose ions C 6 H 13 6 + and the anion of the respective acid. In neutral or alkaline solution, it was to be assumed that glucose as a weak acid would ionize into CeH^Og" and hydrogen ions. The recent results of Mathews and Bunzel 18 are in good harmony with this assumption. The ionization process was found to be hastened considerably by adding OH~ ions, and was altogether suppressed on adding a larger number of H+ ions, whereas the process C 6 H 12 8 + HC1 = C 6 H 13 (V + Cl- was accelerated to a certain degree by acid, and very much depressed in the presence of alkalies. We can easily explain these phenomena on the consideration that in both cases a salt 11 Y. Osaka, Die Birotation der d-Glucose, Zeitschr. f. Physik. Chcmie. vol. 35, p. 661, 1900. 12 Baeyer and Villiger, Ber. Deutsch. Chem. Ges., vol. 34, pp. 2679, 3612, 1901; ibid., vol. 35, pp. 1189, 3013, 1902; cf. H. H. Bunzel and A. P. Mathews, The Mechanism of the Oxydation of Glucose by Bromine in Neutral and Acid Solutions, Journ. Am. Chem. Soc., vol. 31, p. 464, 1909. is Cohen, Ber. Deutsch. Chem. Ges., vol. 35, p. 2673, 1902 ; cf . Bunzel and Mathews, loc. cit. i* Walden, Ber. Deutsch. chem. Ges., vol. 34, p. 4185, 1901; cf. Bunzel and Mathews, loc. cit. is Walker, Ber. Deutsch. chem. Ges., vol. 34, p. 4115, 1901 ; cf . Bunzel and Mathews, loc. cit. iG J. Stieglitz, Studies in Catalysis, Am. Chem. Journal, vol. 39, pp. 29 and 166, 1908. i^ Loc. cit. is Loc. cit; H. H. Bunzel, The Mechanism of the Oxydation of Glucose by Bromine, Journ. Biol. Chem., vol. 7, p. 157, 1909. 124 University of California Publications in Physiology [VOL. 4 formation must take place. From the work of Stieglitz 19 we know that salts of weak acids and bases are ionized to a far greater extent than the respective acids or bases in free state. From the results above it would follow that we have to regard glucose as a true amphoteric electrolyte with predominating acid character. The free acid is, however, only very scarcely ionized and hence highly inactive. Processes in which the negative glucose ion is involved will proceed with comparatively high velocity while reactions in which the positive ion is alone active (the negative ion being suppressed) proceed very slowly. Mathews and Munzel 20 also tried to determine which of these two glucose ions, CgH^Og" and (C 6 H 13 U + ) could be oxydized in the presence of bromine in different concentrations of alkali and acid, and at what rate the respective reactions would proceed. The progress of the oxydation was determined by measuring the rate of disappearance of the bromine. Bunzel 21 in his last 'publication arrives at the conclusion that in absolute neutrality the oxydations of both negative and positive glucose ions proceed simultaneously, but with different velocities, the oxydation of the negative ion naturally being more rapid than the positive ion oxydation. In an acid medium the rate of oxydation is falling off in direct proportion to the concentration of the acid, until enough acid is present to suppress the oxydation of the negative ion entirely. Beyond this point the addition of more acid has no depressing effect. With regard to the results of Kuff 22 and others there is strong evidence that gluconic acid and finally saccharic acid are the main products of this very slow positive ion oxydation. The presence of alkalies, in accordance with what has already been stated, greatly accelerated the negative ion oxydation in Mathew's experiments, until a certain optimal alkalinity was reached, beyond which the increment falls off again. Alkali is, however, unable to suppress the negative ion oxydation entirely. is LOG. cit. 20 Loc. cit. 21 H. H. Bunzel loc. cit. 22 O. Kuff, Zur Darstellung der einbasischen Sauren der Zuckergruppe, Ber. Deutsch. chem. Ges., vol. 32, p. 2273, 1899. 1912] Birckner: Glucose Oxydations 125 Mathews 23 considers the phenomena depending on the ionization (and the amphotheric properties) of the glucose molecule 24 as having considerable biological significance. The true explanation of the rapid oxydation of sugar in the organ- ism is, according to him, not merely its faculty to activate the oxygen, as had been the almost general assumption for several decades, but more particularly its faculty of increasing in some way or other the "active mass" of the sugar itself, i.e., by increasing its dissociation. A simultaneous activation of the oxygen is of course not impossible, but it is emphasized that, no matter whether the sugar is to be acted upon in an alkaline or neutral or even in an acid solution, the first prerequisite of oxydation is always an increased ionization of the reducing sub- stance, an activation of the oxygen possibly (but not necessarily) being effected at the same time. Similar conceptions of this question we find already in the works of Abderhalden 25 and Schade 20 . The reason why glucose does not oxidize rapidly in vitro is to be sought for chiefly in the glucose molecule itself and not in the lack of active oxygen, and, applying this deduction to well-known pathological conditions of the body, Mathews 27 points out "that a failure of living matter to burn glucose is probably not due to the absence of oxydases, properly speaking, but to the probable loss of its power to dissociate the glucose. Under the term of oxydases there have hitherto been confused two classes of substances; one which activates the oxygen; the other, the more important class, which activates by dissociation the reducing substances. The latter are specific, the former not." Although certainly of importance in view of the fact that in nature sugar is being broken down with apparently equal readiness in both alkaline and acid media, it must not be for- gotten that the dissociation process is only the introductory step 23 A. P. Mathews, The Spontaneous Oxydation of the Sugars, Journ. Biol. Chem., vol. 6, p. 19, 1909. 24 Similar relations may with some certainty be assumed to exist also with regard to other sugars. 25 E. Abderhalden, Lehrbuch der Physiologischen Chemie (Berlin, 1906), p. 102. 26 H. Schade, Die Bedeutung der Katalyse fur die Medicin. (Kiel, 1907), p. 126; cf. Lob, Biochem. Zeitschrift, vol. 20, p. 528, 1909. 27 Loc. cit., p. 20. 126 University of California Publications in Physiology [VOL. 4 for the subsequent chemical reaction. The two complex glucose ions must naturally have very little or almost no stability, and are easily broken down into smaller groups of atoms. These in turn may go into more or less stable combinations, and it can a priori not be anticipated in what way the primary glucose ions may determine the direction of these changes. We possess altogether too little knowledge at present of the nature of the transformation products of glucose in those solutions, to obtain much new information from experiments of this kind. After the question of these reaction products, which at present stands in the center of the interest, is worked out more clearly, experi- ments of the type of those to which reference has just been made will undoubtedly be valued much more highly. 3. The Action of Radiant Energy on Glucose (a) The Electrolysis of Sugars. Lob's Theory. It was not until recently that the question of the products of glucose transformations has been properly attacked. The simplest case is presented in the experiments of Neuberg on the electrolysis of pure organic compounds in aqueous solutions. Neuberg 28 is the first one to use the pure substance without the addition of an electrolyte. He subjected a large number of organic compounds in dilute solution to a direct current in a dark vessel, using platinum electrodes. The conductivity of many of these substances, which w r as actually or nearly zero at first, increased after they had been exposed to the current for some time. With pure glucose, for instance, it took thirty-five minutes before the conductivity became measurable. During the course of the experiment Neuberg took small samples from the x liquid and made qualitative tests, any gas formation being neglected. Thus with a 2 per cent solution of glucose in pure distilled water, after exposing to the current for eighteen hours, he obtained the following reactions: 28 c. Neuberg, Elektrosynthesen in der Zuckerreihe, Biochem. Zeitschr., vol. 7, p. 527, 1908; Chemisehe Umwandlungen durch Strahlenarten II, Bio- chem. Zeitschr., vol. 17, p. 270, 1909. 1912] BircJcner: Glucose Oxydations 127 The liquid reduced Fehling's solution almost instantly in the cold. Likewise an alkaline solution of copper acetate was reduced after a short time. Tollens' 29 orcin reaction for pentoses was given but very slightly. The reaction with naphtoresorcin 30 was very strong, indicating the presence of glucuronic acid or of one of its homologes. On adding barium hydrate, a yellow, flaky precipitate of a basic barium salt was formed. With basic lead acetate the liquid gave a precipitate unlike the original solution. The precipitate dissolved in an excess of the lead reagent. With phenylhydrazine the liquid gave at once a tur- bidity in the cold, a sticky oil separating out on standing The filtrate from this oil soon gave crystals of glu- cosazon in the cold, the latter taking rise from the d- glucosone. No formaldehyde nor trioxy-methylene could be detected. The liquid was fermentable by yeast. Quite similar results were obtained with d-fructose, cane- sugar, raffinose and a- and /3-methylglucoside. The di-saccharides and tri-saccharides as well as the glucosides apparently under- went first a hydrolytic cleavage, whereupon the glucose con- stituent was broken down in its usual way. Some of the products had probably arisen not from the hexose directly, but through secondary changes under the influence of the current. On the whole, Neuberg could conclude from these results that the electrolysis of mono-saccharides even in neutral solution is a means of causing chemical transformation of these otherwise 29 B. Tollens, tiber Farbenreactioneu auf Xylose und Arabinose, etc., LieUg's Ann. d. Chemie, vol. 254, p. 329, 1889. so B. Tollens, tiber einen einfachen Nachweis der Glucuronsaure mittels Naphtoresorcin, HC1, and Aether, Ber. Deutsch. cliem. Ges., vol. 41, p. 1788, 1908. 128 University of California Publications in Physiology [VOL. 4 indifferent substances. The main products resulting from these changes are carboxylic acids and the osones of the respective sugars. "While Neuberg carried out these experiments only recently, Walther Lob had devoted himself to similar studies for a number of years previously. Only his last investigation on the elec- trolysis of grape sugar is of very recent date, too, and it may be briefly reviewed in connection with that of Neuberg. The object of Lob's w T ork was primarily to find whether the simplest sugar, HCOH, could be found among the products of electrolysis of glucose. 31 According to some previous experi- ences, such a transformation seemed indeed to be possible. Thus Buchner, Meisenheimer and Schade 32 had already described the formation of formic acid as one of the products of sugar oxyda- tion by hydrogen peroxide ; von Lebedew 33 had observed f or- maldehyde among the products of the action of the cell-free yeast ferment on sugar. Furthermore, the experiments of "Windaus and Knoop, 34 Neuberg, 35 and Bokorny 36 seemed to contain some more or less direct evidence for an intermediary formation of this substance. The work of Walther Lob 37 leaves no doubt that formaldehyde is actually one of the regular products of the electrolysis of grape-sugar. His manner of experimentation, however, differed from the method employed by Neuberg in several points. He used his glucose in 'a solution containing 5 per cent sulphuric acid, and observed the changes 31 For the earlier literature on the electrolysis of glucose see: E. von Lipp- mann, Die Chemie der Zuckerarten (ed. 3, Fr. Vieweg & Sohn, Braunschweig, 1904), p. 373; or C. Neuberg, loc. cit., Biochem. Zeitschr., vol. 17, p. 270, 1909. 32 E. Buchner, J. Meisenheimer and H. Schade, Alkoholische Garung ohne Enzyme, Ber. Deutsch. chem. Ges., vol. 39, p. 4217, 1906. 33 A. von Lebedew, tiber das Auftreten von Formaldehyd bei der Zell- freien Garung, Biochem. Zeitschr., vol. 10, p. 454, 1908. 3-t A. Windaus and F. Knoop, Die uberfiihrung von Traubenzucker in Methylimidazol, Ber. Deutsch. chem. Ges., vol. 38, p. 1166, 1905. 35 C. Neuberg, Depolymerisation der Zuckerarten, Biochem. Zeitschr., vol. 12, p. 337, 1908. 36 Th. Bokorny, tiber die Assimilation des Formaldehyd und die Versuche, dieses Zwichenprodukt der Kohlensaure Assimilation nachzuweisen, Pflilger's Archiv, vol. 125, p. 467, 1908. 37 W. Lob, Zur Kenntnis der Zuckerspaltungen, m-vii, Biochem. Zeit- schr., vol. 17, pp. 132 and 343, 1908; vol. 20, p. 516, 1909; vol. 22, p. 103, 1909; vol. 23, p. 10, 1909; Zeitschr. f. Elektrochem., vol. 16, p. 1, 1910. 1912 ] Birckner: Glucose Oxydations 129 at each pole separately, the one in touch with the sugar always being a lead spiral (cooled by water running inside), while the other, which was separated from the former by a diaphragm, was a platinum electrode. The glucose was used in a solution of not less than 20 per cent. The potential of the current was four to five volts. Lob found as the chief products at either pole for- maldehyde and pentose, showing at the same time that their formation is due not simply to the electrolytic oxydation at the anode, but takes place in the same way, although more slowly, under the reducing influence of the kathodic hydrogen. At the anode a marked secondary oxydation to the corresponding acids was observed. As a consequence of the relatively high inactivity of formaldehyde and pentose, their oxydation takes place far more slowly than that of the glucose itself, whereby the equi- librium C 6 H 12 6 = C 5 H 10 5 -f HCOH is shifted towards the right, resulting in an excess of pentose and aldehyde. These two, according to Lob, are the real dissociation products of glucose, and he assumes an equilibrium of the same type to establish itself in the aqueous solution of every aldose sugar. As already stated, he succeeded in showing that under the influence of the kathodic hydrogen the equilibrium was shifted in the same direction as at the anode, the glucose being far more readily reduced (forming mannite) than its dissociation products at the right side of the formula. Theoretically there was, therefore, no intelligible reason why in Neuberg's experiment no formaldehyde reaction could be obtained. Lob, 38 on repeating the experiment in the manner described by Neuberg, 33 actually could make sure that formalde- hyde is also formed in the electrolysis of glucose in a non- acidulated solution without the use of a diaphragm. 40 For the reaction that takes place at the lead anode in his original experiment, Lob 41 proposes the following formulation : 38 Loc. cit., Biochem. Zeitschr., vol. 22, p. 105, 1909. 39 Loc. cit. 40 For the methods used by Lob for the identification of formaldehyde see Zeitschr. f. Elektrochem., vol. 16, p. 2, 1910. 41 Loc. cit. 130 University of California Publications in Physiology [VOL. 4 Glucose C.H ia Pentose C 5 H 10 5 Formaldehyde HCOH CO, CO 2 , HCOOH Formic acid the first line representing the primary reaction, the second line showing the products of secondary oxydation processes. Lob has shown in a similar way that the same relations do also hold for the transformation of the lower aldoses and their respective alcohols under the influence of the current. At least the formation of formaldehyde was common to all of them. The formation of the next lower aldose, however, could never be detected so far (except when starting from the hexoses), although its initial formation is beyond doubt. There was noticed instead, however, a remarkable tendency for a re-formation of the more stable pentoses from these lower and rather unstable depoly- merization products. Thus, starting from glycerine, for instance. Lob 42 obtained ( 1 ) a fair amount of formaldehyde ; (2) a syrupy mass, free from glycerose, glycolose, dioxyaceton and hexose, but containing a pentose (i-arabinose). Lob regards as the introductory step of this reaction the formation of glycerose by the anodic oxygen (Glycerine -{- = glycerose -f H 2 0), which sugar at once dissociates into glycolose and formaldehyde, the glycolose and glycerose in statu nascendi uniting to form pentose. In analogy to the formulation for glucose, these changes may be represented as follows: 42 Loc. cit., Zeitschr. f. EleJctrochem., vol. 16, p. 5, 1910 ; Biochem. Zeit- schr., vol. 17, p. 343, 1909. 1912] Birckner: Glucose Oxydations 131 Glycerose C 3 H 3 Glycolose C 2 H 4 O 2 Formaldehyde CH 2 HCOOH, CO, CO 2 C 5 H S 7 Trioxy-glutaric acid Through these experiments, Lob has not only illustrated a new way by which hexoses may be changed into pentoses, but he has also established the fact that the synthesis of glucose is a reversible process in strictly chemical sense, and that we have a right to speak of a depolymerization of the hexose molecule. By means of electrolytic methods, this molecule can be caused to give off gradually all those six formaldehyde groups from which it was originally built up. It should be stated here that the experiments just referred to furnished only a confirmation of the remarkable theory that Walther Lob had already worked out previously from results of others and his own. I consider it worth while to say a few words about these theoretical deductions of an author whose work has contributed so much to the better understanding of the mechanics of sugar synthesis and transformations. His theory, fantastic as it may have impressed the reader at first, enables us to look upon these problems from quite a new standpoint, .and may possibly contain the key to important discoveries in the future. The fundamental basis of Lob's theory is the well-known hypothesis of Baeyer, 43 according to w r hich the assimilation of carbon dioxyde through the green parts of the plant takes place essentially in two steps. The C0 2 is first reduced by the action of the chlorophyll to formaldehyde, and the latter then condenses 43 A. von Baeyer, Tiber die Wasserentziehung und ihre Bedeutung f iir das Pflanzenleben und die Canning, Ber. Deutsch. chem. Ges., vol. 3, p. 63, 1870. 132 University of California Publications in Physiology [VOL. 4 to form sugar. The process is frequently expressed by the fol- lowing two chemical equations : CO 2 + H a = H COOH + O a (1) 6 HCOH = C 6 H 12 O 6 (2) This hypothesis has withstood the attacks of four decades, and according to the present views, we possess no other theory that explains the facts observed so well as this. It has lately been confirmed, especially by the results of Losanitsch, 44 Lob, 45 Fenton, 46 and Usher and Priestly. 47 As the correctness of this theory became more and more obvious, W. Lob, 48 extending it to the reversed reaction, con- cluded that formaldehyde is really the central figure in the following three biochemical processes ( 1 ) the synthesis of sugar in the plant ; (2) the respiratory oxydation; (3) the alcoholic fermentation. Artificial syntheses of glucose by purely chemical means, 49 as well as those by silent electric discharge, 30 had shown that formaldehyde is among the intermediary products. Now formaldehyde on account of its highly toxic qualities cannot exist in the tissues as such. It is, furthermore, in its usual state too inactive to combine readily with other radicles. Lob therefore assumes it to be present in a "tautomeric, active," 44 S. M. Losanitsch, tiber die Elektroynthesen, Ber. Deutsch. diem. Ges., vol. 40, p. 4656, 1907. 45 W. Lob, Zur Kenntnis tier Assimilation cler Kohlensaure, Zeitschr. f. Elektrochem., vol. 11, p. 745, 1905; Lanclw. Jahrbiicher, vol. 35, p. 541, 1906; Studien iiber die chemischen Wirkungen der stillen elektrischen Entladung, Zeitschr. f. Elektrochem., vol. 12, p. 282, 1906. 46 H. J. Fenton, The Eeduction of Carbon Dioxide to Formaldehyde in Aqueous Solutions. Journ. Chem. Soc., vol. 91, p. 687, 1907. 4 7 F. L. Usher and J. H. Priestley, The Mechanism of Carbon Assimila- tion in Green Plants, Proc. Boy. Soc. (B), vol. 77, p. 369, 1906; ibid., vol. 78, p. 318, 1906; ibid., vol. 84, p. 101, 1911. 48 W. Lob, Zur chemischen Theorie der Alkoholgarung, Zeitschr. f. Elek- trochem., vol. 13, p. 511, 1907; Zur Geschichte der chemischen Gahrungs- hypothesen, Biochem. Zeitschr., vol. 29, p. 311, 1910, and literature cited on page of this article. 49 Butlerow, E. Fischer, O. Low, Tollens; cf. Neuberg, Biochem. Zeit- schr., vol. 24, p. 152. so Berthelot, Thenard, Brodie, Maquenne, Losanitsch und Jovitschitsch, Butlerow, W. Lob; cf. Neuberg, loc. cit. 1912 J Birckner: Glucose Oxydations 133 form in which it cannot be detected by the usual reactions. By submitting a mixture of alcohol and C0 2 in alkaline solution to the silent electric discharge, Lob also succeeded in proving the intermediate formation of CO and H 2 during this slow synthetic process. As the simplest way of representing his active tauto- meric modification of formaldehyde, I would hence suggest the following equilibrium, COH 2 =(CO + H 2 ), the unstable form, represented by the right side, being almost alone present in the tissues and in reactive mixtures. Only if under certain conditions (for instance, in those artificial syntheses) the rate of transformation becomes abnorm- ally slow, these unstable compounds to the right will accumulate in such quantities as to cause a shifting of the equilibrium towards the left, i.e., a formation of the stable compound. We may therefore regard this unstable combination of a CO and a H 2 group as the "active mass" of the formaldehyde, the for- mation of which must precede every reaction in which formalde- hyde is actually involved, no matter whether a higher aldehyde is being broken down to C0 2 and water, or whether it is being synthesized from them. In both cases the final products are ultimately resulting from some intramolecular rearrangement, either by mutual oxydation or reduction, between the CO and H components of the "active" formaldehyde molecules. Accord- ingly, Lob represents the synthesis of sugar and its reversed processes in the following manner : Sj-nthetic process. 6 CO + 6 H 2 = C 6 H 12 O 6 endotliermic (intramol. reduction) Fermentative process. 6 CO + 6 H 2 =2 C.H.OH + 2 CO, 1 exotllrmic Oxydative process. > (intramol. oxyda- 6 CO + 6 H 2 + 6 O 2 = 6 CO 3 + 6 H,O tion) . The essential course, for instance, of the alcoholic fermenta- tion, according to this scheme, would be the following: The sugar under the influence of zymase dissociates com- pletely into active (tautomeric) formaldehyde groups, the active 134 University of California Publications in Physiology [VOL. 4 components of which recombine by a sort of intramolecular oxydation (eliberating heat) to form alcohol and carbon dioxyde. The first part of the process (complete depolymerization of the sugar molecule into active (tautomeric) formaldehyde groups) is the common precursor of all three reactions quoted above. It is only in the second stage (synthetic phase) that differences occur depending on the respective experimental con- ditions (the presence or absence of oxygen, etc.). The unstable groups CO and H 2 , it is easily seen, may indeed form a great variety of combinations the nature of which will be determined solely by the conditions and the relative stability of the respective compounds. The synthesis of sugar over formaldehyde, either artificially or in nature, is therefore in its main part only a special one out of a number of coordinate reactions, and the same would be true for the synthetic formation of pentoses from glycerine, which process was mentioned above. The term "dissociation products" of glucose, which has been used several times in the text of this section, is perhaps apt to cause some confusion. Lob in fact assumes the existence of an equilibrium in aqueous solution between glucose and its dis- sociation products, which as such would not need to have the properties of true ions/' 1 At least one of the dissociation products must be the unstable form of formaldehyde (CO.H 2 ). The other dissociation product may be a pentose or some lower aldose or ketose. 52 Under certain conditions glucose may be com- pletely dissolved into (CO.H 2 ) groups. Now supposing we have the system C,H 12 0.= (CO.H 2 ) + C S H 10 3 . . It is not to be understood that this equilibrium can be traced chemically. It is in fact under normal conditions so much in favor of the hexose, that no formaldehyde or pentose tests will 51 Similar assumptions are also met with in the paper of J. U. Nef, Dissociationsvorgange in der Zuckergruppe I, Liebig's Ann. d. Chcmie, vol. 357, p. 214, 1907. 52 No distinction needs to be made here between aldose and ketose sugars since they are easily convertible into one another, as we know from the work of Lobry de Bruyn, Ber. Deutscli. chem. Ges., vol. 28, p. 3078, 1895, and of Wohl and Neuberg, ibid., vol. 33, p. 3099, 1900. 1912 ] Birckner: Glucose Oxydations 135 be given. Nor would it be possible by arranging conditions in such a manner as to induce a synthetic formation of sugar from formaldehyde, to obtain any more hexose. Both dissociation products are hence to be assumed as existing in some unstable (potentially active) state, and yet we are able to create con- ditions 53 under which these labil dissociation products become concentrated enough to condense partly into their stable form, whereupon they become detectable by chemical means. This depolymerization process, which helps to loosen the molecule and which is the prerequisite of any sugar oxydation, may possibly be brought about in the living body by some ferment acting on the sugar; at any rate, Lob, like Mathews (loc. cit.) ascribes an inability of the organism to burn sugar to conditions, in the body, which are very unfavorable to the dissociation process. This unique and very original theory of Lob does not rest merely on speculative grounds, but is the result of many years of experimental work. One more instance may follow : Lob 54 boiled a solution of pure formaldehyde with zinc dust, using a reflux condensor. From the products obtained (chiefly polyoxy-acids, volatile ketones, especially methylketol and acetol, and a syrupy sugar residue) it was certain that the first stage of the reaction had been a synthetic formation of sugar. On repeating the experiment with glucose instead of formaldehyde, the products were exactly the same, only in different quantitative proportions (as was to be expected). Lob thus obtained evidence of the fact that the decomposition of boiling glucose under the catalytic influence of zinc forms only a part of the reaction that takes place with formaldehyde under the same conditions. Summing up the main results of Lob 's work we may say : (1) The oxydative disintegration of the sugar mole- cule, under all circumstances, takes place in two different stages : (a) A depolymerization process (splitting off of one or more tautomeric (CO.H 2 ) groups). 5 3 See the experiments of Neuberg and Lob cited in the first part of this section. 54 Biochem. Zeitschr., vol. 12, p. 466. 136 University of California Publications in Physiology [VOL. 4 (6) An oxydation process (synthesis of C0 2 and H 2 O or of C 2 H D OH and C0 2 ). The alcoholic fermentation is therefore essentially a synthetic process. (2) The two stages enumerated under (1) a and b, represent not only the biological but the strictly chemical reversion of the process of sugar synthesis. I may call attention to the notable fact that this theory is at the same time an excellent illustration of the close relationship existing between normal respiration and alcoholic fermentation, which was long predicted by Pfeffer. 55 (b) The Action of Light. Interesting experiments have been carried out lately by Neuberg and P. Mayer on the effect of light on solutions of glucose. The biological importance of both light rays and electric rays has long been known to medicine, and both forms are widely used in therapeutics, especially of recent years. Almost nothing was known, however, about the chemical function of these agents until quite recently. Only few organic substances, indeed, are sensitive enough to undergo spontaneous changes under the influence of light, with a measurable velocity. Catalytic substances must therefore be employed with necessity in the study of these phenomena. Neuberg 56 found the salts of uranium to be very apt for this purpose. He investigated the influence of light on many organic substances in 1 to 5 per cent solutions, w r hich contained from 0.5 to 1 per cent of uranium salt. Most of the samples, which contained the catalysor, showed changes within a short time (a few hours or even minutes) after being exposed to the sun-rays, while the control samples in the dark-room remained unchanged. Corresponding sets, which had been exposed to the light with- out the addition of the catalysor, remained likewise unchanged. The reactions which took place were mostly of an oxydative 55 W. Pfeffer, Pflanzenphysiologie, vol. 1, p. 555, 1897. See also Czapek, Die Atmung d. Pflanzen, Ergebn. d. Physiol., vol. 9, p. 605, 1910. 50 C. Neuberg, Chemische Umwandlungen durch Strahlenarten, I, Bio- chem. Zeitschr., vol. 13, p. 305, 1908. Birckner: Glucose Oxydations 137 character, the hexoses generally being transformed to the respec- tive osones, cZ-glucose also giving a positive pentose reaction with orcin. 57 It could also be observed that the amount of change varied greatly with the intensity of the light, and with light from different sources. That a possible contamination of the uranium salts with traces of radium had nothing to do with their catalytic action was shown in the first place by the fact that the samples in the dark-room underwent no change. . Furthermore, Neuberg ascertained in special experiments that even strong preparations of radium were without influence on his substances. In a later communication, Neuberg 58 describes similar experi- ments with iron salts, which led essentially to the same results. P. Mayer 59 has very recently studied the destruction of glucose by light under the catalytic influences of traces of alkali. He measured the progress of the transformation by the changes in optical rotation and in reducing power. These transforma- tions were not the same as those caused by alkali alone in higher concentration (in which case acids are the main products), but under the influence of the rays of a Heraeus quartz lamp, Mayer observed largely the same products that Neuberg had foun4 in the experiments to which reference has just been made. On the whole, it follows clearly from all these investigations that by using certain simple catalytic agents, many substances which as such have no photochemical qualities, and among which are the common sugars, can undergo rapid transformations when exposed to the light. These changes are a specific function of the light, and, as both observers have found, they are inde- pendent, within wide limits, of variations of temperature. No exact quantitative data are, however, available at present as to the extent of these transformations nor concerning the influence of different factors. We know that owing to its structure the plant is especially susceptible to light-actions. We further know that electric forces 57 Tollens, loc. tit. 58 C. Neuberg, Chem. Umwandl. durch Strahlenarten,, iv, Biochem. Zeit- schr., vol. 29, p. 379, 1910; v, ibid., vol. 39, p. 158, 1912. 59 P. Mayer, tiber Zerstornng des Traubenzuckers dureh Lieht, Biochem. Zeitschr., vol. 32, p. 1, 1911. 138 University of California Publications in Physiology [VOL. 4 are active in the animal body. We may finally take it for granted that the catalytic complements of both forms of radiant energy are normally present in living matter. Hence it is obvious that light rays as well as electric potentials may be reckoned as being among the factors by which in part the oxydative disintegration of organic matter in the body may be brought about. 4. The Action of Alkalies and Acids on Glucose That sugars may be easily decomposed by alkalies 60 is a very old experience. It is, however, only very recently that one has studied the nature of these processes more closely. Thus, the knowledge that these changes can be effected by the alkali as such, i.e., by the hydroxyl ion, is of rather new date. Meisen- heimer 61 was the first to show conclusively that glucose can be decomposed by dilute NaOH with the formation of acids, also in the absence of oxygen. The hydroxyl ion, therefore, not only hastens the dissociation process, but it causes the indifferent molecule to break down into more reactive bodies by some sort of intramolecular rearrangement. This fact at the same time explains readily why Mathews 62 observed far more rapid oxyda- tions with alkaline glucose solutions, which had previously been kept for some time in an atmosphere of hydrogen, than with those that had not been treated that way. Mathews also observed that normally in the presence of air the rate of oxydation was accelerated by alkali but only up to a certain optimal alkalinity (between n and 2n NaOH), beyond which the velocity of oxyda- tion falls off again. Mathew T s ascribes this phenomenon mainly to a hindering effect that the strong alkali apparently exerts on the rate of solution or on the chemical action of the oxygen. oo The mechanism of the interconversion of glucose into mannose and fructose under the influence of alkali, which phenomenon was first observed by Lobry de Bruyn and Van Ekenstein, is not very well under- stood; and as this reaction does not involve a permanent rupture of the molecule or yield a stable oxydation product, its discussion may be omitted in this abstract. (For reference see: Eec. trav. chim. d. Pays-Bus, vol. 14, pp. 156 and 203, 1895.) 61 J. Meisenheimer, tiber das Verhalten von Glucose, Fructose u. Galac- tose gegeniiber verdiinnter Natronlauge, Ber. Deutsch. chem. Ges., vol. 41, p. 1009, 1908. 62 A. P. Mathews, Journ. Biol. Chem., vol. 6, p. 3. 1912 ] Birckner: Glucose Oxydations 139 A considerable amount of study has been devoted to the question whether or not an alkalinity corresponding to that of our blood, when acting on glucose at body temperature, could bring about the destruction of the sugar with some rapidity. Up to very recently this was indeed generally supposed to be the case, as with the old titration methods the alkalinity of the blood was found much higher than it actually is, if only the concentration of the OH" ions is taken into account. For pre- paring solutions of very low alkalinity the use of free alkalies is not yery suitable; but mixtures of monobasic and di-basic phosphates have been used with much advantage. By using such mixtures Michaelis and Rona 63 have shown not long ago that an alkalinity equal to that of human blood 64 causes no measurable destruction of glucose in dilute solution in one day. It would be unjustified, however, to apply this result, the correctness of which as it stands can not be doubted, to physio- logical conditions without certain restrictions, as has really some- times been done. 05 As W. Lob 66 rightly points out, it is very well possible that even the slight alkalinity of the blood, which as such would be practically without action on the sugar, might be very much accelerated in its action by other substances in the blood which may act as catalysors. This point has been overlooked by Michaelis and Rona. Indeed, their results become very different if the solutions be supplied with an ample amount of active oxygen, either by bubbling a current of oxygen gas through the liquid or by adding some H 2 2 . It is well known that active oxygen as well as oxydizing substances are always present in the blood. AVith an alkalinity of less than N/10 NaOH in dilute glucose solution, and in the presence of an oxydizing agent Lob 67 not only observed changes in the sugar content, but was able after twenty hours to determine quantitatively the amount of formic 03 L. Michaelis and P. Bona, Die Alkaliempfindlichkeit des Trauben- zuckers, Biocliem. Zeitschr., vol. 23, p. 364, 1909. 0-4 The concentration of H + ions in blood is 0.3 X 10~ 7 ; that of water at blood temperature, 0.85 X 10~ T . 65 See P. Mayer, loc. cit., p. 2. oo W. Lob, Zur Frage der Glycolyse, I, Biochem. Zeitschr., vol. 29, p. 316, 1910. 07 Biochem. Zeitschr., vol. 23, p. 22, 1909. 140 University of California Publications in Physiology [VOL. 4 acid formed, and besides to obtain qualitative tests for formalde- hyde and pentose. That changes occur under these conditions of very low alkalinity, is also affirmed by the extensive studies of Jolles 68 on the destruction of the different sugars at body tem- perature. Jolles kept all his solutions at a constant alkalinity of N/10 NaOH, by adding fresh alakli in proportion as the original amount became neutralized. The accelerating influence on the acid formation of the addition of hydrogen peroxyde was readily observed. But while according to Lob's last communi- cation 69 oxydizing agents in neutral solutions are without effect at ordinary temperature, Jolles 70 reports quite recently a very different result. In a neutral 2 per cent solution of glucose, to which only hydrogen peroxyde had been added, he was able to prove the formation of glucuronic acid after six days at a temperature of 37 C. in two different cases. Jolles also made complete analyses of his alkaline sugar solutions. Thus, for instance, he found that an n/100 alkaline NaOH) solution of pure dextrose (3 per cent), on standing at 37 C. for almost five months without the addition of an oxydiz- ing agent, contained at the end of this time ethyl alcohol, formic acid, acetic acid, and lactic acid. Unfortunately he does not make any statement as to how the solution was kept sterile for such a long time, a fact which would of course be important to know for the correct interpretation of these results. With regard to the influence of weakly alkaline phosphate mixtures on glucose, W. Lob in his last paper, which I just took occasion to refer to, finds a new fact of importance, namely that in the presence of H 2 0.> the phosphate ion P0~ 4 itself has a dis- tinctly catalytic influence on the action of the OH ions on glucose, which influence is still perceptible if the hydrogen ion concentration of the liquid becomes greater than that of water, i.e., if the fluid alreday shows an acid reaction. This influence of the P0 4 ion is specific to this group, and increases in direct os A. Jolles, Zur Kenntnis des Zerfalls der Zuckerarten, Biochem. Zeit- schr., vol. 29, p. 152, 1910. ca Biochem. Zeitschr., vol. 32, p. 47, 1911. "o A. Jolles, tiber eine neue Bildungsweise der Glucuronsaure, Biochem. Zeitschr., vol. 34, p. 242, 1911. 1912] Birckner: Glucose Oxydations 141 proportion with its concentration, the concentration of the OH" ions being kept constant. This catalytic effect of the phosphate ion can be greatly depressed or even inhibited by the addition of organic derivatives, especially of peptons, proteins, or sera. Of other substances that show an action on glucose by means of OH~ ions which they form in contact with water, those that have been particularly studied are the metals, zinc, lead (Lob, loc. cit.} and potassium. 71 The formation of the nitrogen compound methyl-glyoxaline from glucose by the action of zinc hydroxyde ammonia, at ordinary temperature, as observed by Windaus and Knoop, 72 may also be mentioned in this connection. In turning to the question as to what are the products of the action of alkali on glucose, we have to distinguish between the specific effect of the OH" group as such, and its action as catalyst in sugar oxydations. The product characteristic of the action of OH" groups on glucose in the absence of air is lactic acid. Buchner, Meisen- heimer and Schade 73 from a 2 per cent solution of glucose in n/NaOH in closed vessels recovered as much as 50-60 per cent of the sugar in form of lactic acid 74 after prolonged standing at low temperature. This transformation represents the chemical analogon of the lactic acid fermentation. Nef 73 in his laborious studies found that under the influence of NaOH 7G the main products of sugar decomposition besides the racemic form of lactic acid are a mixture of saccharins C G H 10 5 , 71 J. Stoklasa, tiber die Zuckerabbau fordernde Wirkung des Kaliums, etc., Zeitsclir. f. Physiol. Chemie., vol. 62, p. 47, 1909. 72 A. Windaus and F. Knoop, three articles in Ber. d. Deutsch. chem. Ges., vol. 38, p. 1166; vol. 39, p. 3886; vol. 40, p. 799. 73 E. Buchner, J. Meisenheimer and H. Schade, Zur Vergarung des Zuckers ohne Enzyme, Ber. Deutsch. chem. Ges., vol. 39, p. 4217, 1906. 7 4 The formation of lactic acid from dextrose by the action of alkali had previously been observed by: F. Hoppe-Seyler, Ber. Deutsch. chem. Ges., vol. 4, p. 346, 1871. Kiliani, ibid., vol. 15, p. 701, 1882. Schiitzenberger, Compt. rend., vol. 76, p. 440, 1873. Framm, Pfliiger's Archiv., vol. 64, p. 575, 1896. 75 J. U. Nef, Dissociationsvorgange in der Zuckergruppe, Liebig 's Ami. d. Chem., vol. 357, p. 214, 1907; ibid., vol. 376, p. 1, 1910. 76 Not only of Ca(OH)o (Kiliani, Ber. Deutsch. chem. Ges., vol. 15, p. 2960; vol. 26, p. 1650; vol. 35, p. 3530; cf. Nef, loc. cit. I, p. 303. 142 University of California Publications in Physiology [VOL. 4 a statement the correctness of which was at first doubted by Meisenheimer. 77 Nef also found that the variety of products is much smaller after the action of strong (8n) NaOH than with lower alkalinities, in which latter cases it becomes extremely difficult to separate and define the multitude of products. If oxydizing agents such as metallic oxydes are employed together with the alkali, no saccharin formation takes place (Nef, loc. cit.}. The products in this case are largely polyoxy- acids and aldehydes. As is generally known, the oxydation of sugars by metallic oxydes in alkaline solutions forms the principle of many methods of sugar determinations. The oxydizing agent most commonly used is known under the name of Fehling's solution, a strongly alkaline liquid containing cupric salt and potassium-sodium tartrate. In the presence of aldoses or ketoses the cupric salt on heating is reduced to cuprous salt, the amount of which can be determined either by volumetric or by gravimetric methods. It should be stated that all methods based on this principle are purely empirical. The course of the reaction is unknown, and it has no definite endpoint. The investigator, therefore, is compelled to adhere rigidly to the directions given to him by the man who worked out the respective sugar tables. About forty differently composed Fehling's solutions have so far been devised. The modifications which have been found to give the most satisfactory results, and which are the ones most widely used at present are those of Allihn, 78 Pfliiger, 70 Bertrand, 80 and I. Bang. 81 In connection with Pfliiger 's method, which is fre- quently used in medical laboratories for the determination of 77 J. Meisenheimer, loc. cit. 78 Allihn, tiber den Verzuckerungsprocess bei der Einwirkung von verdiinnter Schwefelsaure auf Starkemehl bei hoherer Temperatur, Journ. f. prakt. Chemie, N.F., vol. 22, p. 46, 1880. For two other articles see v. Lippmann, Chemie der Zuckerarten (Ed. 3; Braunschweig, Fr. Vieweg und Sohn, 1904), p. 591. 79 E. Pfliiger, tiber eine neue Methode zur quantit. Bestimmung des Zuckers, etc., Pfliiger 's Arch. f. d. ges. Physiol., vol. 66, p. 635. Unter- suchungen iiber die quantitative Analyse des Traubenzuckers, ibid., vol. 69, p. 399; also later volumes up to vol. 129, p. 362, 1909. so G. Bertrand, Le dosage des sucres reducteurs, Bull. Soc. Chim., vol. 35, p. 1285, 1906. si Ivar Bang, Zur Methodik der Zuckerbestimmung, Biochcm. Zeitschr., vol. 2, p. 271, 1906. 1912 ] Birckner: Glucose Oxydations 143 the glycogen content of animal organs, 82 it may be well to call attention to a frequent source of error in such determinations caused by the iron content of the respective organs, as was lately pointed out by Starkenstein. 83 Nef s latest view concerning the nature of the products which arise from hexoses under the influence of Fehling's solution, may be found in an article of his pupil Anderson. 84 Griefenhagen, Konig and Scholl, 85 in an interesting investi- gation, have lately worked out a new method of sugar deter- mination, which is based on the observation that oxalic acid is a regular oxidation product of all sugars, if they be acted upon by KMnO 4 and alkali, and that its formation takes place quan- titatively. If 'the sugar is a hexose, the process was found to follow the equation C 6 H 12 O 6 + 5 O 2 = 2 H 2 C 2 O 4 + 2 CO, + 2 H 2 O. By using a standard solution of KMn0 4 at the start, the amount of oxygen given off gives an exact measure of the amount of sugar present. The method so far as investigated gave very satisfactory results. The oxydation products of glucose in the presence of the halogen elements or of acids are chiefly those that show no rupture of the original carbon chain, viz., glucomc acid and saccharic acid. The formation of glucuronic acid under the influence of H 2 2 8C has already been mentioned. Together with phenoles in strongly acid solutions, glucose, as is generally known to be the case with all carbohydrates, gives rise to colored compounds, chiefly furfurol derivatives. 82 See E. Pfluger, Das Glycogen (2te. Aufl., Bonn. 1905), p. 106; Meine Methode der quantitativen Analyse des Glycogens, etc., Pfluger 's Archiv, vol. 129, p. 362, 1909. 83 G. Starkenstein, uber den Glyeogenhalt der Tunicaten, nebst Ver- suchen uber die Bedeutung des Eisens fiir die quantitative Glycogen- bestimmung, Biochem. Zeitschr., vol. 27, p. 53, 1910. s* E. Anderson, On the Action of Fehling's solution on Galatose, Am. Chem. Journ., vol. 42, esp. pp. 403-406. 85 W. Greifenhagen, J. Konig und A. Scholl, Bestimmung der Kohle- hydrate durch Oxydation mittels Kaliumpermanganat in alkalischer Lb'sung, Biochem. Zeitschr., vol. 35, p. It59, 1911. so See Jolles, loc. cit. 144 University of California Publications in Physiology [VOL. 4 H. Schade 87 has much endeavored to find chemical analoga of fermentative reactions. According to his studies, it is possible by a successive application of the catalytic influences of alkali, acid, and metal, to bring about an alcoholic fermentation of glucose by purely chemical means. He represents the different stages of the process in the following way : Dextrose (Alkali as catalysor) Lactic acid (Sulphuric acid as catalysor) Acetic aldehyde + formic acid (Ehodium as catalysor) Alcohol + COo B. THE OXYDATTONS AND CLEAVAGES OF GLUCOSE THROUGH THE ACTION OF MORE OR LESS UNKNOWN AGENCIES The phenomena to which reference will be made in this sec- tion are largely of a nature which at the present state of our knowledge w r e have no means of explaining in a satisfactory way. There is is a strong tendency to attribute most of these phenomena to some sort of fermentative activity. But although this assumption has a certain degree of probability in view of the fact that ferment action may be met with outside of as well as inside of the organism, we know of only comparatively few cases in which the presence of a ferment has been demonstrated convincingly. The oxydative destruction of glucose may either be complete or incomplete. A complete cleavage leads to the formation of CO 2 , and w r e may include under this heading the processes in- volved in respiration phenomena (both normal and intra- molecular respiration) and the alcoholic fermentation. As an incomplete oxydation we may regard the formation of acids. This is, of course, only an artificial classification; no sharp line can really be drawn between complete and incomplete sugar oxydations. s? H. Schade, tiber die Vorgange der Garung vom Standpunkt der Katalyse, BiocJiem. Zeitschr., vol. 7, p. 299, 1907. 1912 ] Birckner: Glucose Oxydations 145 The process which is known best, and which has been studied for the longest time, is that of alcoholic fermentation. Nobody will doubt any longer the fermentative character of this process. Only the nature of the intermediary stages of the reaction is not yet sufficiently elucidated. That the intramolecular respiration of living organisms, as far as it concerns the sugar, is in all chemical respects identical with the alcoholic fermentation was predicted by Pasteur 88 as early as 1872. It was definitely proven by the more recent investigations of Godlewski and Polszeniusz, 89 Stoklasa, 00 Palladin and Kostytschew, 91 and Maximow. 92 As far as the mechanism of the normal respiration is con- cerned, we may say that we are still in the very beginning of this study. That ferments are involved in these processes, however probable, has not at all been sufficiently proven. Up to recently it was widely assumed that the oxygen is activated by some oxygen catalyst in the respiratory organs. At present, however, it is believed that the more important prerequisite of sugar combustion is a far-reaching dissociation or loosening of the hexose molecule itself. Whichever may be the real point of attack, it has become customary to speak of this sugar trans- formation as of an act of glucolysis, and the hypothetic ferment, which is assumed to form the active principle in these changes, has been termed the glucolytic ferment of the body. Very little is known about the individuality and the mode of action of this ss L. Pasteur, Note au sujet d 'une assertion de M. Fremy, etc., Compt. rend., vol. 75, p. 1056, 1872. 89 Godlewski und Polszeniusz, tiber intramoleculare Atmung (Krakau, 1901); cf. Czapek, Ergeb. der Physiologic, vol. 9, p. 606, 1910. 90 J. Stoklasa, Hofm. Beitrdge zur chem. Physiologic und Path., vol. 3, p. 460, 1902; Zentralbl. f. Physiologic, vol. 16, p. 652, 1902; Ber. Deutsch. chem. Ges., vol. 36, p. 622, 1903; ibid., p. 4058; Zentralbl. f. Bacteriologie, Abt. II, vol. 13, p. 86, 1904; Ber. Deutsch. Bot. Ges., vol. 22, p. 460, 1904; Pfliiger's Archiv, vol. 101, p. 311, 1904; Zeitschr. f. physiol. Chemie, vol. 49, p. 303,^1907; Festschrift fiir Wiesner, p. 218, 1908; Zeitschr. f. Zucker- industrie Bohmens, vol. 32, p. 273, 1908; cf. Czapek, Ergeb. d. Physiol., vol. 9, p. 589, 1910. si W. Palladin und S. Kostytschew, Anaerobe Atmung, Alokoholgarung und Acetonbildung bei den Samenpflanzen, Zeitsclir. f. Physiol. Chemie, vol. 48, p. 214, 1906; Ber. Deutsch. Bot. Ges., vol. 24, p. 273, 1906. 2 N. A. Maximow, Zur Frage liber die Atmung. Ber. Deutsch. Bot Ges vol. 22, p. 225, 1904. 146 University of California Publications in Physiology [VOL. 4 ferment, and its very existence has not infrequently been actually questioned. 83 The incomplete oxydation of glucose to acids is chiefly known as a vital function of many microbes. That these organisms act by certain ferments, which they form in their body, is widely assumed, but only in a very few cases, so far, could a respective glucolytic ferment be isolated. For the lactic acid formation in muscle, the interaction of a ferment is expressively denied by Fletcher and Hopkins 94 in their recent communications. I shall at first dwell upon a number of cases in which a destruction of glucose was effected in organic fluids by some agent or agents of unknown character, other than cellular activity. Finally, I shall give a brief outline of the sugar transformations that are known to take place in living organisms. The discovery of Buchner 95 that the sugar-splitting prin- ciple can be extracted from the yeast, and that it can transform glucose into alcohol and C0 2 outside of the cell is so well known in- its details and in its bearing on our present conceptions of fermentation problems that I can refrain, in this review, of giving a complete description of this fundamental observation. 96 The next example, to my knowledge, of a destruction of sugar in plant extracts, was furnished by M. Hahn 97 in 1900. Halm studied the autodigestion of a juice, which he had obtained by crushing the flowering shafts of Arum maculatum by means of a hydraulic press in the manner described by Buchner (loc. cit.) for yeast. On standing at room temperature the liquid showed a rapid decrease in sugar content, accompanied by a decrease in weight and an ample production of C0 2 . The reaction was 93 See Abderhalden, Lehrbuch der Physiologischen Chemie (2 te Anil. Urban & Schwarzenberg, Berlin, 1909), pp. 107, 596. 9* W. M. Fletcher and F. G. Hopkins, Lactic acid in amphibian muscle, Journ. Physiol., vol. 35, p. 247, 1906; W. M. Fletcher, On the alleged for- mation of lactic acid in muscle during autolysis and in post-survival periods, Journ. Physiol., vol. 43, p. 286, 1911. 95 E. Buchner, Alkoholische Garung ohne Hefezellen, Ber. Deutsch. chem. Ges., vol. 30, pp. 117, 1110, 1897; ibid., vol. 31, p. 568, 1898. 6 For method see Buchner und Hahn, Die Zymasegarung, Miinchen, 1903. See also the much more simple method of zymase preparation recently devised by von Lebedew (Comp. rendus, vol. 152, pp. 49, 1129, 1911. Zeitschr. f. Physiol. Chemie, vol. 73, p. 447, 1911). 97 Martin Hahn, Chemische Vorgange im Zellfreien Gewebsaft von Arum maculatum, Ber. Deutsch. chem. Ges., vol. 33, p. 3555, 1900. 1912 J Birckner: Glucose Oxydations 147 greatly depressed at 60 C. and was arrested after boiling. Hahn ascribed these phenomena to the presence of an oxydative ferment. In turning to the animal kingdom, we meet with numerous researches for glucolytic ferments, especially in the blood. Scheremetjewski 98 had already observed that in blood, which contained sugar, the oxygen content decreased on standing, while the C0 2 content increased correspondingly. Later on, Cl. Ber- nard 09 stated that from fresh blood, on standing, the sugar dis- appears, and that its place is taken by lactic acid. 100 Lepine 101 confirmed this statement and ascribed the phenomenon to the action of a glucolytic ferment. Krauss 102 also assumed such a ferment, but at the same time pointed out that it could not play an important part in sugar metabolism as its small efficiency is in no proportion to the great quantities of sugar that are con- stantly undergoing change. Rohmann 103 and Spitzer 104 observed similar decompositions of sugar not only in the blood, but in aqueous extractions of many organs. They considered this phenomenon, however, as a function of the living protoplasm at that time. The experiments of N. Sieber 105 showed the presence of three glucolytic ferments in the fibrine of the blood plasm. The three fractions could be obtained as powders, and showed some of the typical reactions of the oxydases. This author, therefore, is inclined to consider the oxydases as of great bio- 8 Scheremetjewski, tiber die Anderung des respiratorischen Gasaus- tausches durch d. Zufiigung verbrennlicher Molecule zum kreisenden Blute, Sachs. Ges. Wissensch. Math. phys. Kl. (Leipzig, 1868), vol. 20, p. 154. 99 Claude Bernard, Legons sur le diabete (Paris, 1877), p. 128. 100 On the latter statement cf. T. Launder Brunton, On a probable glycolytic ferment in muscle, etc., Zeitschr. f. Biologie, vol. 34, p. 487, 1896. 101 R. Lepine, several articles in Comptes rendus de I'Acad. des Sciences, 1890-1895; cf. Abderhalden, Lehrbuch der Physiologischen Chemie, 2 te. Aufl., 1909, p. 89. 102 F. Krauss, Zeitschr. f. klinische Medisin, vol. 21, p. 315, 1892; cf. N. Sieber, Zeitschr. f. Physiol. Chemie, vol. 39, p. 507, 1903. 103 Zentralblatt. f. mediz. Wissenschaften, vol. 51, p. 849, 1893; cf. Sieber, loc. cit. 104 Berliner klinische Wochenschrift, 1894, p. 949; cf. Sieber, loc. cit. 105 N. Sieber, Einwirkung der Oxydationsenzyme auf Kohlehydrate, Zeitschr. f. Physiol. Chemie, Vol. 39, 484, 1903; Zur Frage nach dem glyeolytischen Princip des Blutfibrins, idem., vol. 44, p. 560, 1905. 148 University of California Publications in Physiology [VOL. 4 logical importance. The progress of the reaction was measured, 3is in previous investigations, by the disappearing of the sugar and the simultaneous formation of C0 2 Of the three fractions, the alcohol soluble part especially showed a marked resistence against heat. For muscular tissues, Claude Bernard 106 showed that not only do dead muscles become acid at the expense of the sugar and glycogen they contain, but that they also cause the formation of acid in a solution of grape-sugar to which they are added. Brunton (loc. cit.) is the first, in a brief note, to mention a glucolytic ferment of the muscle. Cohnheim 107 succeeded in extracting the inactive form of this ferment, and showed that it could be activated by a co-ferment which he obtained from an alcoholic extraction of the pancreas. 108 Cohnheim chiefly studied the relation between the amount of co-ferment added, and the extent of the sugar, destruction. He noticed the formation of small quantities of acids, including slight amounts of C0 2 gas, leaving, however, the question of the products open for its main part. Levene and Meyer, 100 in a recent attempt to clear up this point, failed to detect carbonic, formic, acetic or lactic acids among the products resulting from this apparent disappearance of glucose. In their belief, the latter phenomenon is due to a condensation process (formation of a di-saccharide) under the influence of the combined tissue extracts. Sieber and Dzierzgowski 110 claim that the cell-free liquid that 100 Cf . Brunton, loc. cit. 107 Cohnheim, Die Kohlehydrateverbremmng in den Muskeln, und ihre Beeinflussung durch das Pancreas, Zeitschr. f. Physiol. Chemie, vol.*39, p. 336, 1903; ibid., vol. 42, p. 401, 1904; tiber Glycolyse, ibid., vol. 47, p. 253, 1906. !os Stoklasa's recent investigations (J. Stoklasa, tiber die glucoly- tischen Enzyme im Pancreas, Zeitschr. f. Physiol. Chemie, vol. 62, p. 36, 1909) have definitely shown that from the pancreas itself (pig's pancreas) no ferment can be extracted, that would have some action on hexoses. See also G. W. Hall, Concerning Glycolysis, Am. Journ. Physiol., vol. 18, p. 283, 1907. io P. A. Levene and G. M. Meyer, Journ. Biol. Chem., vol. 9, p. 97, 1911; ibid., vol. 11, p. 347, 1912. no N. Sieber und W. Dzierzgowski, Die Enzyme der Lunge, Zeitschr. f. Physiol. Chemie, vol. 62, p. 263, 1909. 1912 ] Birckner: Glucose Oxydations 149 can be obtained by crushing the lungs of horses, even after previously washing out all the blood, contains, among others, a glucolytic ferment, while Levene and Meyer 111 report a different result with the tissues of other animals. Finally, I may mention an observation made by Vandevelde. 112 If to normal urine glucose was added, and the mixture kept at 37 C. under sterile conditions, a distinct fall of the optical activity was observed after one year's standing, while the reducing power against Fehling's solution remained unaltered. The same observation w r as made with diabetic urine (no addition of sugar). The phenomenon, I may infer, is perhaps due to the formation of an inactive combination product between glucose and urea. 113 All these investigations which are confined to the measure- ment of the disappearance of sugar or of the consumption of oxygen in connection with a production of C0 2 , do, however, not approach the real problem itself. We know sufficiently well that sugar is constantly being broken down in the organism, and that C0 2 and water, or C0 2 and alcohol, respectively, are the end-products of these transformations. The real problem, is, however, to find out by what means the living substance brings about these rapid transformations, or, as the first step towards this aim, to find the intermediary stages in these oxydations. C0 2 , and even alcohol may be readily formed, according to recent investigations, from dead organic materials, 114 even from nitrogen containing substances, 115 either spontaneously or through the action of yeast (the latter case referring to the alcoholic fer- 111 P. A. Levene and G. M. Meyer, Journ. Biol. Chem., vol. 11, p. 353, 1912. 112 A. J. J. Vandevelde, Polarimetrisch messbare Zuckerzerstorungen in physiologischen Fliissigheiten, Biochem. Zeitschr., vol. 23, p. 324, 1909. us See M. N. Schoorl, Les ureides (earbamides) des sucres, Eec. des trav. des Pays-Bas et de la Belg., vol. 22, p. 1, 1903; also P. Mayer, tiber Ureidoglucose, Biochem. Zeitschr., vol. 17, p. 145, 1909. 114 See F. Czapek, Ergeb. d. Physiol., vol. 9, p. 600, 1910, and references on that page. us See F. Ehrlich, tiber eine Methode zur Spaltung racemischer Amino- sauren mittels Hefe, Biochem. Zeitschr., vol. 1, p. 8, 1906; ibid., vol. 8, p. 438, 1908; also O. E. Ashdown and J. T. Hewitt, The by-produets of alcoholic fermentation, Journ. Chem. Soc., vol. 97, p. 1636, 1910; O. Neu- bauer and K. Fromherz, tiber den Abbau der Aminosauren bei der Hefe- garung, Zeitschr., f. Physiol. Chemei, vol. 70, p. 326, 1910; C. Neuberg and others, tiber Zuckerfreie Hefegarungen, Biochem. Zeitschr., vol. 31, p. 170, 1911; ibid., vol. 32, p. 323, 1911; ibid., vol. 36, p. 60, 1911. 150 University of California Publications in Physiology [VOL. 4 mentation of nitrogen compounds), so that the observations to which I have referred above do not give us any essentially new information. As to the intermediary stages of the alcoholic fermentation, our conceptions have undergone various changes in recent years. Very likely we shall have to encounter two or more intermediate products in this process, resulting from two or more coordinate reactions which proceed independently of one another and each of which is catalyzed by a different constituent of the zymase preparation. Although the assumption of an intermediate formation of lactic acid 110 is now generally abandoned, 117 the formation of a substance closely related to it is very probable. 118 Certain yeasts have the faculty of producing, during the normal fermentation process, marked amounts of formic acid, and this process is regarded as an intermediary step of the alcoholic fermentation by some authors. 119 Furthermore, the intermediate formation of ester-like com- binations between glucose (and perhaps also between a resulting triose) and phosphate groups seems to be a very important factor in the process of alcoholic fermentation, and phosphoric acid in us E. Buchner und J. Meisenheimer, Die chemischen Vorgange bei der alkoholischen Garung, Ber. Deutsch. diem. Ges., vol. 37, p. 419, 1904; ibid., vol. 38, p. 620, 1905. A. Wohl, Die neueren Ansichten iiber den chemischen Verlauf der Garung, Blocliem. Zeitschr., vol. 5, p. 45, 1907. 117 E. Buchner und J. Meisenheimer, Die chemischen Vorgange bei der alcoholischen Garung, iv, Ber. Deutsch. chem. Ges., vol. 43, p. 1773, 1910. us P. Boysen Jensen, Die Zersetzung des Zuckers wiihrend des Kes- pirationsprocesses, Ber. Deutsch. Bot. Ges., vol. 26a, p. 666, 1908. Sukker- s0nderdelingen under Eespirationsprocessen hos H0jere Planter, Dissert. Copenhagen, 1910. us See for instance: H. Franzen, Tiber die Vergarung und Bildung der Ameisensaure durch Hefen, Zeitschr. f. Physiol. Chemie, vol. 77, p. 129, 1912. 1912 J Birckner: Glucose Oxydations 151 organic combination (e.g., as lecithin) is looked upon as the activating complement of zymase. 130 Recently, Harden and Young, 121 von Lebedew, 122 and still later Euler and his coworkers, 123 have studied more closely the structure of these hexose phosphates, and the manner in which they are hydrolyzed again. Harden and Young represent this phosphate cycle of the alcoholic fermentation in the following way : (I) 2 C,H M 0. + 2 R' 2 HP0 4 > 2 C0 2 + 2 C 2 H 6 O + C 6 H 10 O 4 (PO 4 E',) 2 + H 2 0. (II) C 8 H 10 4 (P0 4 R' 2 ) 2 + 2 tt,0 > C 6 H 12 6 + 2 R' 2 HPO 4 . Reaction (II), according to these authors, is brought about by a special enzyme which they call "hexose phosphatase. " This whole formulation is strongly opposed, however, by von Lebedew (loc. cit.}. According to Kostytschew and Scheloumow, 124 the stimulating effect of the phosphate salt is perhaps due only to its alkaline reaction. 120 Numerous articles by: A. Harden and W. J. Young, Proc. Chem. Soc., vol. 21, p. 189, 1905; ibid., vol. 24, p. 115, 1908; Soy. Soc. Proc. (B), vol. 77, p. 405, 1906; ibid., vol. 80, p. 299, 1908; ibid., vol. 81, p. 336, 1909; ibid., vol. 82, p. 321, 1910; Centralbl. f. Bacterial., (n) vol. 26, p. 178, 1910; Biochem. Zeitschr., vol. 32, p. 173, 1911. W. J. Young. Proc. Chem. Soc., vol. 23, p. 65, 1907; Roy. Soc. Proc. (B), vol. 81, p. 528, 1909; Biochem. Zeitschr., vol. 32, p. 177, 1911. H. Euler und G. Lundeqvist, Zeitschr. f. Physiol. Chemie, vol. 72, p. 97, 1911. L. Iwanoff, Trav. Soc. des Natur. de St. Petersbourg, vol. 34, 1905, cf. Euler und Ohlsen, below; Zeitschr. f. Physiol. Chemie, vol. 50, p. 281, 1906; Centralbl. f. Bacterial, (n), vol. 24, p. 1, 1909; Biochem. Zeitschr., vol. 25, p. 171, 1910. E. Buchner und J. Meisenheimer, loc. cit. E. Buchner und W. Albertoni, Zeitschr. f. Physiol. Chemie, vol. 46, p. 136, 1905. 121 Biochem. Zeitschr., vol. 32, p. 173; and references quoted sub W. J. Young. 122 A. von Lebedew, Versuche zur Aufklarung des zellfreien Garungs- processes mit Hilfe des Ultrafilters, Biochem. Zeitschr., vol. 20, p. 114, 1909; Tiber Hexosephosphorsaureester, Biochem. Zeitschr., vol. 28, p. 213, 19lO;ibid., vol. 36, p. 248, 1911. 12 s H. Euler und A. Fodor, fiber ein Zwischenprodukt der alcoholischen Garung, Biochem. Zeitschr., vol. 36, p. 401, 1911; H. Euler and H. Ohlsen, Tiber den Einfluss der Temperatur auf die Wirkung der Phosphatese, ibid., vol. 37, p. 133, 1911. 124 S. Kostytschew und A. Scheloumow, Tiber die Einwirkung der Garungsprodukte und der Phosphate auf die Pflanzentamung, Jahrb. f. wiss. Botanik, vol. 50, p. 157, 1911. 152 University of California Publications in Physiology [VOL. 4 Boysen Jensen (loc. cit.) obtained evidence for the presence of two forms of dioxyacetone, an isomeride of lactic acid, in the glucose-zymase digest under certain experimental conditions. He regards dioxyacetone as a regular intermediate product of both the alcoholic fermentation and the normal respiration, in accordance with the following scheme : (Dextrase) (Oxydase) Dextrose > Dioxyacetone > CO 2 + H 2 O C0 2 + C 2 H 5 OH The ferment zymase, it is seen, consists of the two fractions dextrase and dioxyacetonase. Besides, after the arrival at the dioxyacetone stage, an oxdyase may come into action (oxydases are not known to attack either glucose or alcohol). We may therefore imagine that, depending on whether the respective organism contains primarily the ferment oxydase or the ferment dioxyacetonase, the second half of the reaction will either be an act of normal respiration, or an alcoholic fermen- tation. Higher organisms have largely lost the power of form- ing dioxyacetonase, and therefore their subsistence depends on the presence of oxygen, while in some lower organisms, with high content of dioxyacetonase as compared with the oxydase, the alcoholic fermentation is predominant. Many of the lower fungi (e.g., yeasts) have the faculty of forming both ferments simultaneously, and in their life, the normal respiration and the alcoholic fermentation proceed side by side. Others (e.g., Mucor, and even phaenogamic plants and lower animals), which normally are consumers of atmospheric oxygen, may become producers of alcohol in case of any shortage in the oxygen supply (phenomena of intramolecular respiration). Boysen Jensen has been able to prove the formation of dioxyacetone directly. It is also known that dioxyacetone is readily ferment- 1912] Birckner: Glucose Oxydations 153 able, 125 while the other three substances which were temporarily regarded as intermediary products (lactic acid, methyl gly- oxaline, and glycerose), are not, or only difficultly (glycerose), fermented by yeasts. 126 W. Lob 12T finds Jensen's results in agreement with his own theory. Apart from these cases, the instances in which products of fermentative sugar oxydations have been obtained, other than CO 2 , H 2 0, and alcohol, are very few in number. Buchner and Meisenheimer 128 have ascertained the presence of a ferment in Bac. Delbriicki (syn. Bac. acidificans longissimus} which is able to split glucose into two molecules of dl-lactic acid. Weinland 129 demonstrated the presence of an enzyme in the clear "press- juice" of Ascaris lumbicoides which splits glucose into valerianic acid, carbon dioxyde, and hydrogen. W. Lob 130 succeeded in showing that the alcohol-soluble part of pig's pancreas, if brought into a firm combination with iron, will act on glucose in a way similar to the action of peroxydases. As among the products of this glucolysis, he obtained small amounts of C0 2 , formic acid, formaldehyde (traces), and pentose. I have myself succeeded in obtaining a ferment preparation from yeast, which shows considerable glucolytic activity, prefer- ably at an elevated temperature. It causes no gas formation and furnishes a solid, carbon-like substance as a result of pro- longed action. Among the products are acids, but I have also been able to ascertain the formation of formaldehyde and pen- 125 G. Bertrand, Etude biochimique de la bacterie du sorbose, Ann. de chim. et de phys., (8), vol. 3, p. 187, 1904; Buchner and Meisenheimer, Ber. Deutsch. chem. Ges., vol. 43, p. 1773, 1910. See, however, also A. Slator, Ber. Deutsch. chem. Ges., vol. 45, p. 43, 1912. 120 Cf. from E. L. Pinner, Fortschritte der Garungschemie, Fortschr. der Chemie, Physik und Physical. Chemie, vol. 4, p. 135, 1911. IK Biochem. Zeitschr., vol. 29, p. 311, 1910. 128 E. Buchner und J. Meisenheimer, Enzyme bei Spaltpilzgarungen, Ber. Deutsch. chem. Ges., vol. 36, p. 634, 1903; tiber die Milchsauregarung, Liebig's Ann. d. Chemie, vol. 349, p. 125, 1906. 120 E. Weinland, tiber Kohlehydratzersetzung ohne Sauerstoffaufnahme bei Ascaris, einen tierischen Garungsprocess, Zeitschr. f. Biologic, vol. 42, p. 55, 1901; tiber ausgepresste Extrakte von Ascaris lumbicoides und ihre Wirkung, ibid., vol. 43, p. 86, 1902; tiber die von Asc. lumbicoides aus- geschiedene Fettsaure, ibid., vol. 45, p. 113, 1903. iso \v. Lob und Pulvermacher, Biochem. Zeitschr., vol. 29, p. 316, 1910. 154 University of California Publications in Physiology [ V OL. 4 tose. A report of the work is given in the second part of this paper. While all the authors so far quoted in this section considered these glucolytic ferments as being of the general type of oxydases, Euler 131 objects to this classification. According to him the oxydase reactions, which these preparations mostly give at the same time, have no relations to their glucolytic qualities. As the latter function is of primary importance, and as in this respect, all these ferments resemble Buchner's zymase, Euler unites all glucolytic ferments under the heading "Garungs- enzyme," separating them from both the oxydases and the hydrolytic ferments. Little more needs to be said about the cleavages of glucose in living organisms. Glucose in the animal body is the main source of muscular and of respiratory energy. The places in which its oxydation transformation is going on, for its main part, are the blood, the lungs, and the muscles. Apart from the fact that the final products of these transformations are C0 2 and H 2 0, their chemical nature is very little understood. In vitro, we are familiar with three principal types of sugar disintegrations, namely, (1) Direct Oxydations (without cleavages of the mole- cule ; resulting in the formation of gluconic and saccharic acids) ; (2) Depolymerizations (splitting off of one or more formaldehyde groups. Reversion of photosynthesis) ; (3) Cleavages (resulting in the formation of lactic acid or its homologes. Theoretically, each of these three types might be involved in the sugar combustion in living tissues, as by means of secondary processes C0 2 and H 2 (or alcohol) could easily be the final products of each of these reactions. As far as we know, true cleavage processes resulting in the intermediary formation of lactic acid are of predominating importance in the sugar meta- bolism of animals. Levene and Meyer 132 have just found that 131 Allgemeine Chemie der Enzyme, p. 37. 132 p. A. Levene and G. M. Meyer, Journ. Biol. Chem., vol. 11, p. 361, 1912. 1912 J Birckner: Glucose Oxydations 155 the leucocytes of the blood play a very prominent part in these transformations. Grlucuronic acid, which in combinations forms a constituent of many organs and tissue fluids, is perhaps derived from glucose by an oxydative process the nature of which is presently unknown. We possess a far more extensive knowledge, however, of the metabolic sugar cleavages in lower plants, and in bacteria. Here again we would have to mention yeasts in the first place, the study of which is a constant source of valuable information. 133 The genetic relationship between the alcoholic fermentation (anaerob respiration) and the respiration of higher organisms with regard to glucose was early recognized by Pfeffer, 134 and has been clearly established through the newer researches of Godlewski, 135 Palladin, 130 Kostytschew, 137 Walther Lob, 138 Zaleski, 13 " and Boysen Jensen. 140 For an excellent review of the 133 For the kinetics of alcoholic fermentation, see M. J. H. Aberson, La fermentation alcoolique, Eec. d. trav. chim. des Pays-Bas, vol. 22, p. 78, 1903, and H. Euler, Chemische Dynamik der Zellfreien Garung, Zeitschr. f. Physiol. Chemie, vol. 44, p. 53, 1905. 134 \v. Pfeffer, Das Wesen und die Bedeutung der Atmung, Land- wirtschaftl. Jahrbiicher, vol. 7, p. 805, 1878. 135 E. Godlewski, Bull, intern, de I'Acad. des Sc. de Cracovie, 1904, p. 115; cf. Euler, Pflanzenchemie, vol. 2, p. 172, 1909. See below. i3u \y. Palladin, tiber den verschiedenen Ursprung der wahrend der Atmung d. Pflanzen ausgeschiedenen Kohlensaure, Ber. Deutsch. Bot. Ges., vol. 23, p. 240, 1905; Bildung d. verschiedenen Atmungsenzyme in Abhan- gigkeit von dem Entwicklungsstadium der Pflanzen, ibid., vol. 24, p. 97, 1906; tiber das Wesen der Pflanzenatmung, Biochem. Zeitschr., vol. 18, p. 151, 1909. i3T S. Kostytschew, tiber die Alkoholgarung von Aspergillus niger, Ber. Deutsch. Bot. Ges., vol. 25, p. 44, 1907; Zur Frage der Wasserstoffbildung bei der Atmung d. Pflanzen, ibid., p. 178; uber anaerobe Atmung ohne Alkoholbildung, ibid,, p. 188; Zweite Mitt, uber anaerobe Atmung ohne Alkoholbildgun, ibid., vol. 26a, p. 167, 1908; tiber den Zusammenhang der Sauerstoffatmung der Samenpflanzen mit der alkoholischen Garung, ibid., p. 565; tiber die Anteilnahme der Zymase am Atmungsprozesse der Samen- pflanzen, Biochem. Zeitschr., vol. 15, p. 164, 1908; tiber den Vorgang der Zuckeroxydation bei der Pflanzenatmung, Zeitschr. f. Physiol. Chemie, vol. 67, p. 116, 1910; besides, together with W. Palladin, Zeitschr. f. Physiol. Chemie, vol. 48, p. 214, 1906. 138 LOC. dt. 1 39 W. Zaleski, Zum Studium der Atmungsenzyme der Pflanzen, Bio- chem. Zeitschr., vol. 31, p. 195, 1911; W. Zaleski und A. Reinhard, Unter- suchungen iiber die Atmung der Pflanzen, Biochem. Zeitschr., vol. 35 p. 228, 1911. 1*0 Loc. cit. 156 University of California Publications in Physiology [VOL. 4 recent advances along these lines we are indebted to Euler. 141 It remains to enumerate briefly the action on glucose of some of the more important micro-organisms. One of the typical oxydizing bacteria is the Bacterium xylinum (Adrian Brown), which is more commonly known from the studies of Bertrand 142 as the "sorbose bacterium." Besides other activities, this microbe is able to oxydize glucose to gluconic acid. This fermentation was first observed by Bou- troux, 143 who called the microbe Micrococcus oblongus. All other vital fermentations, so far as known, result in a cleavage of the glucose molecule. We have already referred to the important group of bacteria that split glucose into two molecules of lactic acid. These microbes are of general occur- rence in most organic food materials, especially in milk and certain beverages derived from it, such as the well-known "kefir" or the Bulgarian "yoghurt." 144 They have also been found to be active in the stomach of higher animals in cases of carcinoma. 145 That the living leucocytes of the blood have the power of breaking down glucose into d-lactic acid is one of the latest results obtained by Levene and Meyer. 146 Besides, there have been observed organisms which effect the formation from glucose of the following substances. 141 H. Euler, Grundlagen und Ergebnisse der Pflanzenchemie (Braunsch- weig, Fr. Vieweg u. Sohn, 1909) 2nd and 3d part, p, 171 and following. 142 G. Bertrand, Action de la bacterie du sorbose, etc., Compt. rend. r vol. 126, p. 984, 1898; ibid., vol. 127, p. 124, 1898; La bacterie du Sorbose, Ann. Chim. Phys. (8), vol. 3, p. 181, 1904. 1*3 L. Boutroux, Sur une fermentation nouvelle du glucose, Compt. rend vol. 91, p. 236, 1880. !44 See, for instance, G. Bertrand und F. Duchacek, tiber die Einwirkung des Bacillus bulqaricus auf verschiedene Zuckerarten, Biochem. Zeitsclir., vol. 20, p. 100, 1909. 145 Sandberg, Zeitschr. f. klin. Medisin., vol. 51, p. 80, 1903; cf. Emmer- ling, Biochem. Centralbl., vol. 9, p. 408, 1909. i4c p. A. Levene and G. M. Meyer, Journ. Biol. Chem., vol. 11, p. 361 > 1912. 1912] Birckner: Glucose Oxydations 157 Fermentation products Oxalic acid Citric acid Acetic acid Succinie acid Propionic acid Butyric acid Propyl-alcohol n-butyl-alcohol Butylene-glycol Acetyl-methylcarbinol See for reference : W. Zopf , Ber. Deutsch. Bot. Ges., vol. 18, p. 32, 1900. G. Wehmer, Bot. Zeitung, 1891, p. 233. G. Wehmer, ChemiJcer Ztg., 1897, p. 1022. P. Maze and A. Perrier, Ann. de I'Inst. Pasteur, vol. 18, p. 553, 1904. E. Buchner and Wiistenfeld, Biochem. Zeitschr., vol. 17, p. 395, 1909. Gayon and Dubourg, Ann. de. I'Inst. Pasteur, vol. 15, p. 527, 1901. O. Emmerling, Ber. Deutsch. chem. Ges., vol. 37, p. 3535, 1904. Harden and Walpole, Proc. Boy. Soc. London (B), vol. 77, pp. 399, 519, 1907; vol. 83, p. 272, 1911. For more complete references to this paragraph see : O. Emmerling, Die Zersetzung stickstofffreier organischer Sub- stanzen durch Bakterien (Braunschweig, Fr. Vieweg, 1902); Neuere Arbeiten auf dem Gebiet der Bakteriengarungen, Biochemisches Zentralblatt, vol. 9, p. 397, 1909. E. von Lippmann, Die Chemie der Zuckerarten (Ed. 3, Braun- schweig, Fr. Vieweg & Sohn, 1904), p. 374 and following. 158 University of California Publications in Physiology [Voi,. 4 PART II YEAST GLUCASE, A NEW GLUCOLYTIC FERMENT On the preceding pages I have at several points alluded to a fermentation phenomenon which I first happened to observe some time ago, and to the study of which I have lately devoted considerable time. A preliminary report on this work may follow : THE FERMENT AS FIRST OBSERVED AND RECOGNIZED In the early part of 1911, following a suggestion of Dr. T. Brailsford Robertson, I found it necessary for a certain purpose to prepare the ferment maltase. I tried to obtain it from yeast by the well-known method of Croft Hill 1 or with a slight modification according to 0. Emmerling. 2 The material chosen was the yeast of the so-called California "steam beer," a local brew, which although a bottom fermentation beer, differs in many respects from the common lager beers. Steam beer originated (in San Jose) soon after the discovery of gold in California, and as its characteristic quality is rapidity of preparation, we may infer that it was intended chiefly for the purpose of meeting the strong demands of those "early days." Up to about twenty years ago, steam beer was practically the only beer produced on this coast. The differences between lager beer and steam beer depend in the first place on the difference in temperature at which the fermentation of the wort is carried on, and they are therefore 1 A. Croft Hill, Reversible zymo-hydrolysis, Journ. Cliem. Soc., vol. 73, p. 634, 1898. For the method see also Euler, Allegmenie Cliemie der Enzyme (Wiesbaden, 1910), p. 15. 2 0. Emmerling, Synthetische Wirkung der Hefemaltase, Ber. Deutsch. chem. Ges. vol. 34, p. 602. Birckner: Yeast Glucase 159 a direct function of the respective metabolic activity of the yeast. The following table will serve to illustrate these relations : Temp, at start Temp, maximum which is allowed Time required for the whole ferm. process 5C. 10-11 C. 8 to 10 days 13 C. 18 C. 3 days Lager beer California ' ' Steam ; Besides the temperature, it is probably the more extensive aeriation of the steam-beer yeast which in part causes its high activity. After having reached the temperature of 18 C., instead of being cooled down by artificial means in the fermenting vat itself, the whole brew is transferred to large wooden pans, the so-called "clarifiers," where in a layer about one foot deep the fermentation process is carried to the end. Although in this way, by giving the mixture a large surface, the rise of temperature is checked, the fermentation process still proceeds with considerable speed on account of the ample aeriation. The use of these "clarifiers" is a characteristic feature in the manufacture of the California steam beer. The yeast of the steam beer has accommodated itself to these conditions to such an extent that it can no longer be employed for the preparation of lager beer, while lager-beer yeast may without difficulty be used for the manufacture of steam beer. The cells of the typical steam-beer yeast are somewhat smaller than those of lager-beer yeast. The yeast used for the most part in the experiments which I am about to describe was furnished by the California Brewing Company of San Francisco. I am highly indebted to Mr. G. Woehrle of this firm, who very kindly supplied me with the necessary material at many occasions. After following the directions of Hill (loc. cit.) and Emmer- ling (loc. cit.) in every detail, I arrived at the conclusion, after numerous trials, that with my particular material (California 160 University of California Publications in Physiology [Voi.. 4 steam-beer yeast) it was impossible to obtain an active maltase prepartion by the method of Hill. 3 The following are only a few of the many negative results. Two mixtures were prepared, each containing 90 c.c of a 5 per cent solution of pure maltose (Kahlbaum), 20 c.c. yeast extract (according to Hill), and 1 c.c. of toluol. The solutions, in tightly stoppered vessels, were placed in incubators, one at a constant temperature of 30 C., the other at 70 C. From time to time, samples of 10 c.c. were removed with a pipette and transferred to a 200 c.c. graduated flask. After making up to volume with distilled water, the optical rotation was determined. The polariscope used was a Schmidt and Haensch triple field nicol-prism instrument, which allowed readings to be made within a hundredth of a degree. Using a 400 m.m. tube and white light, the following readings were obtained. Time in hours Polariscope reading 30 C. 70 C. 1.10 1.10 14 1.12 1.10 42 1.10 1.10 88 1.06 1.04 162 1.10 1.05 Part of the yeast extracted was placed in the 70 incubator for one day. A whiteish precipitate had formed at the bottom. From the supernatant clear liquid, after cooling, 20 c.c. were 3 It is not at all surprising that, in working with living material, methods that have been found useful for a certain operation in one locality may not be successful if employed at a different locality. Ex- periences of that sort have been met much more frequently perhaps than could be concluded from the literature alone. The lower forms of life, such as yeasts and bacteria, are especially sensitive to slight variations of external conditions. Such changes must necessarily affect the one-cell organism much more deeply in its whole structure and organization than they would influence higher forms, where those variations may affect only the function of one special organ. It is of interest in this connection that with yeast material of this locality I am not the first one to report a complete failure of a method that is generally found successful in other places. Taylor (A. E. Taylor, On Fermentation, Univ. Calif. Publ. Pathol., vol. 1, p. 212) after many unsuccessful trials to obtain by the Buchner method (see H. Euler, loc. cit., p. 38) an active zymase preparation from San Francisco yeast, is led to the statement that "the commercial Sticcharomyces cerevisiae of this city is worthless for the preparation of a yeast powder: the glycogen content is high, the proteolytic ferment active, the zymase weak." 1912 1 Birckner: Yeast Glucase 161 tested against maltose in exactly the same manner as described. The result was the following: Time in hours Polariscope reading 30 C. 70 C. 1.10 1.10 14 1.10 1.10 41 1.10 1.09 71 1.07 1.07 143 1.08 1.07 Likewise the white precipitate alone was tested against maltose with the following result : Time in hours Polariscope reading 30 C. 70 C. 1.31 1.31 14 1.31 1.31 41 1.30 1.31 90 1.28 1.28 In all three cases, it is seen, there was practically no activity against maltase. At the time when I still had some hope of having maltase in my solution, part of the extract w r as tried on a strong solution of glucose, on which maltase, according to Croft Hill (loc. cit.) and Emmerling (loc. cit.), exerts a synthetic action, yielding a di-saccharide. I noticed very peculiar changes to take place. After stand- ing in the 70 incubator for one day, the sample showed a reddish brown coloration, which, after two or three days, had changed into dark crimson, the liquid, which was still perfectly clear, having acquired an aromatic odor. An acid reaction against litmus paper w r as also observed. After prolonged stand- ing in the 70 incubator in tightly stoppered test tubes, the samples turned almost black, and a brownish carbon-like sub- stance settled out along the edges of the glass very gradually. No gas formation was noticed. The samples which had been kept in the 30 incubator under- went no visible changes for a very long time, after which they finally became faintly yellow. They, too, gradually gave an acid reaction. The fact that a solution of glucose alone in the same con- 162 University of California Publications in Physiology [VOL. 4 centration did not undergo any similar changes could be easily ascertained. Nor did either maltose or the ferment extract, or both together, show any such colorations for at least two weeks. It was therefore natural to assume that a catalytic agent of some kind was bringing about these changes in the glucose-extract mixture. That the substance which causes the crimson coloration is actually formed at the temperature of 70 C. and not simply in the time during which the mixture is gradually being heated up to this point, was shown by placing each of the two solutions (glucose and extract) in the 70 incubator separately at first, and mixing them when warm. The coloration appeared in the same way as before. On transferring some of the colorless samples, which had been kept in the 30 incubator for weeks, to the 70 incubator, the change in color took place readily, and it could be rendered twice as intense by boiling the sample for a moment just before transferring it. The respective compound, therefore, had pos- sibly been formed at the lower temperature too, and only assumed that different color at the elevated temperature. At any rate, it seemed important to know what was the nature of the substance which causes these characteristic transformations with such regularity. I was at first inclined to think that really a synthetic change from glucose into isomaltose had taken place, an assumption which seemed well justified with regard to what is known about the properties of this di-saccharide. 4 Very soon, however, I could convince myself that this conclusion was erroneous. All di-saccharides are known to have a higher optical rotation and a lower copper reducing power than d-glucose. In bme preliminary experiments I had already noticed, however, that there was taking place in my mixtures not only a decrease in reducing power, but also a decrease in optical activity. These results had been obtained with a ferment preparation that was more than two months old; they therefore needed confirmation. "With a fresh preparation the result was largely the same, as we shall see presently. 4 See E. v. Lippmann, loc. cit., p. 1513. 1912] Birckner: Yeast Glucase 163 To 160 c.c. of a 40 per cent solution of pure glucose (Kahl- baum), 40 c.c. of yeast extract and a few drops of toluol were added, the liquid well mixed, and measured from a burette into test tubes in portions of exactly 10 c.c. The tubes were tightly stoppered and divided into two sets. One set was placed in an incubator at 30 C., the other into the 70 incubator. From time to time, one test tube of each set was taken for analysis, its contents being washed into a one-litre graduated flask. After adding 10 c.c. of a 2.5 per cent solution of sodium carbonate to stop the reaction, the liquid was made up to mark with distilled water, filtered, if necessary, and an aliquot used for the deter- mination of the reducing power, while the remainder was available for the polariscope reading. The liquid had to be used in such a high dilution on account of the coloration, and because it is not advisable, with white light, to obtain readings that would exceed the value of three degrees. 5 The white light of a 50-candle power globe was used with the instrument, as I could not procure a sodium light of sufficient intensity, The variations in temperature were slight, and their influence on the reading was negligible in this preliminary work. The read- ings refer to a tube length of 400 m.m. The determinations of the reducing power were all made by the method of Bertrand (loc. cit.). In the tables below, which are intended to give an approximate idea of the progress of the reaction, only the number of cubic centimeters of the standard KMn0 4 solution which is used in this method have been inserted. Each cubic centimeter is equivalent to 8.72 mg. Cu. The mixture at the beginning was composed of 40 c.c. yeast extract, 80 c.c. glucose (40%) and toluol. (10 c.c. were made up to 1 litre for each determination.) Rotation Polariscope reading 30 C. 70 C. Time in hours Reduction No. of c.c. KMnO 4 required 30 C. 70 C. 0. ,60 0, .60 11, .62 c.c. 11, 62 c.c. 0. ,57 0, ,48 64 11 , 70 c.c. 11, 55 c.c. 0, ,57 0, ,45 110 11 . 65 c.c. 11, , 35 c.c. .53 0, .42 190 11 .58 c.c. 11. 25 c.c. .54 .41 254 11 . 65 c.c. 11 ,15 c.c. 5 See Landolt-Long, The Optical Eotating Power of Organic Substances (Easton, Pa., The Chemical Publishing Co., 1902), p. 417. 164 University of California Publications in Physiology [VOL. 4 The yeast extract, as already mentioned, gave a precipitate in the 70 incubator if kept there for several hours. Both frac- tions, the clear fluid and the precipitate, were tested separately for their action on glucose. The precipitate showed no action whatever, while the clear liquid had preserved its fermentative qualities almost unweakened. In the following table the com- position of the mixture was exactly the same as in the preceding one, except that for the ordinary yeast extract this clear liquid, which has just been described, was substituted : Rotation Time Reduction Polariscope reading in No. of c.c. KMnO 4 required 30 C. 70 C. hours 30 C. 70 C. 0.55 0.55 11. 63 c.c. 11. 63 c.c. 0.53 0.48 63% 11. 58 c.c. 11. 60 c.c. 0.54 0.42 1*234 11. 60 c.c. 11. 35 c.c. From both these tables it follows clearly that there is no synthetic process going on, but that sugar is being broken down. On these figures alone we could, however, not base a reasonable interpretation either of the rate of the reaction or of its products, for the following reasons : Although highly diluted for the analysis, the color of the liquid deepens to such an extent as the reaction proceeds that this color alone causes a marked depression of the polariscope reading, so that the actual loss of rotatory power is probably not nearly so great as would appear from the tables. On the other hand, the fermentation products may be optically active substances themselves, so that in no case would the polariscope reading give an accurate idea of what transformations are really taking place. Hence, although from the last two tables it seems as if the fall in optical activity proceeds much faster than the fall of the reducing power, we can not at all be sure that such is really the case. The destruction of glucose may, on the other hand, yield substances which themselves have a high reducing power against Fehling's solution. Hence this method of determination might be just as inaccurate and misleading in its results as the optical method. 1912] Birckner: Yeast Glucase 165 I therefore had no accurate means of measuring the progress of the reaction, nor was there much chance of finding out, within a short time, what the products of transformation would be. Moreover, as long as I was following the method of Hill, I only could prepare a small quantity of yeast extract at a time. My particular material dried only very slowly in the vacuum over sulphuric acid; and had to be spread in very thin layers, thus giving only small yields. Fortunately, while in these difficulties, I happened to gain a new aspect of the problem by observing that the ferment showed a certain activity against hydroquinone, hastening its oxydation to quinone. Hence it appeared to be a typical oxydase. 6 I therefore decided to prepare a larger quantity of it, and if pos- sible to find a somewhat simpler method of preparation. The new yeast material was procured from a larger brewery this time, which was located not far from the one previously mentioned. This new yeast contained slightly less bacteria (only two per 1000 yeast cells) than that of the California Brewing Company at that time. Both the former as well as the latter were, however, typical steam-beer yeasts. I prepared the yeast powder in three different ways : (1) By following the directions of Hill. (2) By treating the yeast with acetone and ether. (3) By treating the yeast with methyl alcohol and ether. My expectations that this material would yield the ferment in a very pure and active condition were not fulfilled. In fact I lost nearly two months' time in preparing the three different powders, extracting them, and testing the extracts against different solutions of sugar and hydroquinone with improved methods. The final conclusion which I had to draw was that this yeast does not contain a sufficient amount of the ferment in o Griiss and Issajew had already described the presence of an oxydase in yeast (Griiss, Tiber Oxydaseersehienungen der Hefe, Woschenschr. f. Brauerei, vol. 18, pp. 310, 335, 1901; cf. Kastle, The Oxydases, etc., Hyg. Lab. U. S. Pub. Health and Marine-Hasp. Serv., Bull. 59, Washington, 1910; W. Issajew, liber die Hefe-oxydase, Zeitschr. f. Physiol. Chemie, vol. 42, p. 132, 1904.). 166 University of California Publications in Physiology [VOL. 4 question, to be used with advantage for the purpose of its study. Although the presence of the ferment in the extract was beyond doubt, the degree of activity was in all cases only a small fraction of that displayed by the former yeast preparations. As the reasons for this unexpected behavior I finally recog- nized the following facts : It is well known that, as a rule, ferment preparations are rapidly destroyed in aqueous solution by spontaneous hydrolysis under the influence of antibodies (proteolytic ferments), which are mostly quite active at ordinary temperature. The applica- tion of low temperature usually tends to increase the stability of the ferment, by depressing this autohydrolytic process. Accordingly, I had placed some of my clear extracts on ice at several occasions. After a few days these samples showed strong turbidity and a decreased fermentative activity. On the other hand, one of my first preparations, which had been stand- ing together with toluol in broad daylight in a rather warm place for fully six months, had remained clear and had preserved almost its full activity against glucose. The stability of this particular ferment is therefore not favored by low temperature. Now, the reason that the yeast of the California Brewing Com- pany contains this ferment in such appreciable amounts, is very likely to be sought in the fact that this brewery does not possess an ice-plant. During the time when the yeast is not in action, it is hung up in sacks in the fermentation room, which occupies the second floor of the building, being well aeriated but not artificially cooled. In the larger establishment, however, from which I obtained my last working material, the steam-beer yeast, in the same way as the lager-beer yeast, is kept in a cellar cooled by ice. As there are no differences in the treatment of the jteast during its fermenting action at the two different places, it is most probably the use of this refrigerator to which my ill success was due with the last preparations. It is obvious, from what has been said, w y hy this yeast does not develop this particular ferment but in very small amounts. Naturally under these circumstances I had to return to the use of the original yeast material. The first aim, as already stated, was to find out which way of preparing the yeast powder Birckner: Yeast Glucase 167 would be the one that has the least obnoxious effects on the ferment. The experiments with those weak extracts, mentioned in the preceding paragraph, unsatisfactory as they were on the whole, had shown with fair certainty that the acetone yeast as well as the methyl alcohol yeast (Dauerhefe) on extracting yield fer- ment preparations which are in every respect equivalent, if not superior, to those prepared by Hill's method. They had shown, moreover, that the ferment in question is most likely not zymase, as zymase is rapidly destroyed by methyl alcohol. 7 They had finally shown another not unimportant fact. One of the powders, namely the CH 3 OH powder, happened to be already neutral to litmus paper in its aqueous suspension, while the others, being acid, were neutralized with dilute Na 2 C0 3 , when first brought into contact with water. Now I observed that the extract, which was already neutral by itself, not only gave prac- tically no precipitate at 70 C., but together with glucose, the mixture kept much clearer in appearance than in the samples which contained the neutralized extracts, notwithstanding the coloration process. As, besides, the ferment did not seem to be very sensitive to a slightly acid reaction of the medium (it was naturally formed in a medium of pronounced acidity), I have in all subsequent cases avoided the addition of alkali. By thoroughly washing the yeast all the acid can be removed, so that the resulting powder is, on extracting, practically neutral to litmus. Above all, however, the tedious method of Hill (loc. cit.) could now be discarded entirely. A new batch of yeast was all prepared as "Dauerhefe." Three different fixing agents were tried once more, however, in order to determine definitely which of them could finally be used with the greatest advantage. The yeast was at first washed three times with tap water by decantation in glass vessels, after thorough stirring at each application of water. Then the same was repeated twice with distilled water, Avhereupon the thick suspension was transferred to a large Buchner funnel, and the liquid removed by suction. 7 E. Buehner, H. Buchner und M. Hahn, Die Zymasegarung, 1903 ; cf . W. Zaleski, Biochem. Zeitschr., vol. 31, p. 195, 1911. 168 University of California Publications in Physiology [VOL. 4 Now the compressed yeast was divided into three portions. The first was treated with pure acetone, stirred thoroughly, and the suspension poured on a Buchner funnel, and the acetone removed with the air-pump as fast as possible. The wet mass was now replaced in a dish, stirred with pure ether, and the latter again removed by suction. Then acetone was applied for a second time, followed again by ether, and the resulting powder was dried in the air. The process is essentially the same as that employed by Herzog and Saladin. 8 Exactly the same procedure was observed with a second portion of the compressed yeast, except that instead of acetone strong methyl alcohol was used; and likewise with the third portion, for which ethyl alcohol was the fixing agent. The dried powder of each portion was kept in a glass-stoppered vessel, and part of it removed and ground in a mortar whenever an extraction was to be made. The two alcohol preparations represent white, dust-like powders, while the acetone preparation is heavier, and of a somewhat sandy character. On extraction with water, all except the CH 3 OH preparation, which reacts slightly acid, are neutral to litmus paper. The CH 3 OH suspension settles quickly and is easy to filter, while the other two, especially the acetone preparation, settle very slowly, and are very difficult to filter. All three preparations, after passing through a Chamberland filter by means of a pressure pump, were not used directly, but were submitted to a process of repeated precipitations with alcohol and re-dissolutions in water, a process which I shall describe later in the section on purification methods. The fer- ment was finally obtained in the form of a crisp powder which was partly dried in the vacuum after washing with alcohol, partly after additional washing with ether. To my knowledge ether has never been used so far in this connection. But as the evaporation of the alcohol is a slow process, even in the vacuum over sulphuric acid, I tried the possible effect that washing with ether might have on the ferment. s B. O. Herzog und O. Saladin, tiber die Veranderungen der fermenta- tiven Eigenschaften welche die Hefezellen bei der Abtotung mit Aceton erleideu, Zeitschr. f. Physiol. Chemie, vol. 73, p. 263, 1911. 1912] Birckner: Yeast Glucase 169 Finally, a small portion of each of the three raw extracts was slowly boiled for six minutes with animal charcoal 9 and filtered before being precipitated. All these different portions were now tested for their action on hydroquinone. The apparatus employed for this purpose was the same, in principle, as that described by Euler. 10 I have, however, applied a few alterations which, I believe, assure a higher accuracy of the readings without complicating the method as such. Thus, instead of a burette I used a thin glass tube of 100 cm. length, to which I attached a measuring scale, in order to secure a smaller cross-section. The capacity of the full length of the tube (100cm.) was only 17.72 c.c. As I did not have a vessel of the kind described by Euler (loc. cit.) I used an ordinary wide-mouth bottle of rather strong glass, having an opening of 3.7 cm. in diameter and a capacity of about 500 c.c. Inside of this bottle was first placed a small weighing flask into which the ferment solution was measured with a pipette. Then the respective amount of hydroquinone was introduced into the larger bottle, and the latter closed with a tightly fitting rubber stopper which contained two glass tubes, one connecting with the gasometer, the other provided with a glass stop-cock, as outlet for the air. After the bottle had been filled with oxygen and the stop-cock closed, the level of the mercury was read on the scale (bringing the mercury in both branches of the glass tube to the same level), then the small weighing flask tipped over, and the bottle put on the shaking machine, which was already in motion. The motor was arranged so as to give the shaker just about ninety movements a minute, which was found sufficient to accelerate the reaction notably. A reading was taken every minute for half an hour. The temperature was 18 C. in all experiments. The total amount of liquid in each case was 50 c.c. and it was always 0.2 normal with regard to hydroquinone. Equal amounts of the respective ferment solutions of equal strength (0.2 g. purified ferment powder for each case) were 9 Specially purified with HC1; see Euler, as below. 10 H. Euler and I. Bolin, Zur Kenntnis biologiseh wichtiger Oxydationen, Zeitschr. f. Physiol. Cliemie, vol. 57, p. 80, 1908; tiber die chemische Zusam- mensetzung und die biologisehe Eolle einer Oxydase, Zeitschr. f. PhysiJc. Cliemie, Arrhenius Jubelband (69), p. 187, 1909. 170 University of California Publications in Physiology [VOL. 4 employed in all experiments, so as to make possible an exact comparison between the different preparations. The amount of oxygen absorbed, as expressed by the rising of the mercury in the tube, gave a measure of the respective rate of oxydation. "What interests us here is chiefly the total amount of oxygen absorbed in half an hour by the hydroquinone solution under the influence of the different ferment preparations. The follow- ing table expresses these amounts as measured by the respective total differences in centimeters in the height of the mercury level in the tube. The corresponding average oxygen absorption of 50 c.c. of a 0.2 normal solution of hydroquinone alone was 25 cm. on the scale, corresponding to 4.43 c.c. of oxygen. Previous treatment of Nature of fixing agent : ferment extract Methyl alcohol Ethyl alcohol Acetone Final washing with alcohol 42.2cm. 54.0cm. 55.0cm. Final washing with ether 47.8cm. 52.0cm. 63.0cm. Boiled for 6 minutes 16.4cm. 26.0cm. 24.4cm. For all three preparations the influence of the ferment was therefore readily noticeable. The process under the influence of its action proceeded with at least twice its original velocity. No alkali was added in any case. The washing with ether, it is noticed, had a favorable rather than an obnoxious effect on the ferment. Boiling for six minutes had destroyed the fermenta- tive power entirely. Compared with the others, the acetone preparation proved to be the most active against hydroquinone. I may add that in some qualitative experiments I convinced myself that the addition of a trace of MnS0 4 greatly accelerated the action of the ferment on hydroquinone, and that it also accelerated appreciably its action on glucose. Hence the*fer- ment in this respect resembled Bertrand's laccase. 11 On trying the three different preparations on glucose, I found that their relative activity against this substance was not simply n G. Bertrand, Sur le pouvoir oxydant des sels manganeux et sur la constitution chimique de la laccase, Bull. soc. chim. (3), vol. 17, p. 753, 1897; Sur 1 'action oxydante des sels manganeux et sur la constitution chimique des oxydases, Compt. rend., vol. 124, p. 1355, 1897; Sur 1 'inter- vention du manganese dans les oxydations provoquees par la laccase, ibid., p. 1032. 1912 ] Birckner: Yeast Glucase 171 in parallel with their respective activity on hydroquinone. 12 While against hydroquinone the extract of the acetone powder caused the strongest reaction, this extract was the weakest when acting on glucose, judging from the coloration. Both alcohol preparations acted much stronger in the latter case. But ethyl alcohol seemed to be even slightly in advance of the methyl alcohol preparation. This result, in consequence, furnished the standard method for the preparation of the*y eas t powder. The following is the way in which I proceed. METHOD OF PREPARING THE YEAST POWDER The yeast, after removing from the clarifiers (see above) is allowed to drip in the brewery for about two days, so as to become fairly dry. About fifty pounds may now be taken to the laboratory. The mass is at once distributed in several large glass tanks, so as to fill not more than one-fifth of their content. Then while stirring, 13 a stream of tap water is turned in, and the vessel filled to the edge. After the mass has settled, the liquid is siphoned off, and new water added while stirring. This is repeated a third time with tap water, and, after this, twice with distilled water. Great care should be exercised in this work in order to remove all the acid and to secure a neutral reaction of the resulting powder. After the last washing, the yeast settle- ment is poured on big porcelain funnels (Buchner form, the bottom of which consists of porous clay), and the liquid drained off by suction. The sticky compressed mass is now, in portions, placed in a large porcelain dish with absolute alcohol and stirred (best with a clean hand). The suspension is poured back on the funnel and the alcohol removed by suction. The mass is now again placed in a dish, and treated in the same way with ether. The process is then repeated, once with alcohol and once with ether, care being taken that the substance does not stay in con- tact with these liquids longer than necessary. After the second 12 This is in agreement with Euler's remark, concerning the nomen- clature of the glucolytic ferments, to which I have referred in part I, page 154. !3 I usually use the fingers of one hand (well cleaned) as the most effective means of crushing all the small clods of yeast. 172 University of California Publications in Physiology [VOL. 4 treatment with ether, the whitish mass is spread on filter paper until dry. The powder is to be kept in closed vessels in a warm and dry place. METHOD OP EXTRACTING THE FERMENT The method finally adopted for getting an active ferment preparation is the following: A portion of the yeast powder is ground in a mortar to a fine dust. A weighed portion of this is placed in a bottle and thoroughly shaken with ten times its weight of distilled water and about 0.5 per cent of toluol. The bottle is stoppered and kept at a temperature of between 30 and 40 C., or at room temperature. At the end of thirty-six hours the bottle is trans- ferred to the 70 incubator for another thirty-six hours. During this whole period of extraction the bottle is shaken from time to time. At the end of the second thirty-six hours the contents of the bottle are filtered, at first through paper, and then through a small porcelain funnel with clay bottom, using suction. The dark yellow, strongly opalescent filtrate contains the ferment. It may be kept in solution indefinitely, if saturated with toluol, and placed at room temperature. No precipitation will occur. Any addition of water, however, during or after the filtration process, should be avoided, as a strong autodigestion of the extract may set in on dilution. ATTEMPTS AT FURTHER PURIFICATION Although the raw extract showed a distinct activity on both glucose and hydroquinone, the progress of the reaction on glucose, to judge from the coloration, seemed to be rather slew, considering the usual rapidity of fermentative accelerations. I had noticed at the beginning that the yeast gave a strong reaction with iodine, indicating great richness in glycogen. Apparently the extract contained also a good many gummy substances; and it seemed not improbable that by using some kind of a purifica- tion process, I might be able to eliminate part of these substances. At first the method of repeatedly precipitating the ferment with alcohol and redissolving in water was tried, a method which 1912] Birckner: Yeast Glucase 173 has frequently been used, especially when working with oxydases. I followed closely the method described by Euler 14 for the Medicago laccase. The freshly prepared ferment extract was poured into three times its volume of 98 per cent alcohol, the precipitate collected on a hardened filter and dried in the vacuum over sulphuric acid. When dry, it formed a brownish, gluey mass. This was now redissolved in water, filtered, 15 and precipitated again by pouring the filtrate slowly into three times its volume of absolute alcohol. The precipitate was again collected on a filter, washed with alcohol and dried. This manipulation was repeated a third time. But even after this the resulting substance was not a white powder, as Euler 's Medicago laccase, but a very brittle porcelain-like mass, showing that there are still a good many gummy substances contained in it. The process can be somewhat improved by using ether for washing the precipitate, in which case the operation of drying is hastened considerably, while the ether, as can be seen from the table on page 170 above, has no injurious effect on the ferment. The final purification product, if dissolved in water and filtered, is a colorless, opalescent fluid, which contains the fer- ment. It was with preparations of this kind that the hydro- quinone experiments 16 were carried out. Later on, I observed, however, that against glucose these preparations showed far less activity than the original raw extracts. Another method of purification was tried in the following : A. Wurtz 17 in preparing the ferment papain from Carica Papaya, had found that this ferment, although a protein in character, is not precipitated by basic lead acetate. He made use of this observation in purifying his ferment by precipitating most of the impurities with lead. He actually could obtain from his lead filtrate a very active ferment preparation. The same method was used shortly afterwards by 0. Loew 18 for the i* Zeitschr. f. Physilc. Chemie, vol. 69, p. 190, 1909. is There is always a part left which does not redissolve, upon which fact the purification process is based. IB See above, p. 170. IT A. Wurtz, Sur la Papaine, Contribution a 1 'histoire des ferments solubles, Compt. rend., vol. 90, p. 1379, 1880. is Oscar Loew, "fiber die chemische Natur der ungeformten Fermente, Pfliiger's Archiv. f. ges. Physiol, vol. 27, p. 203, 1882. 174 University of California Publications in Physiology [VOL. 4 purification of diastase, apparently with good success. Euler, 19 in referring to Loew's work, misrepresents this method by stating that Loew had set free his ferment from the lead precipitate. At any rate, it seemed worth while to try this convenient method with my ferment. I followed closely the directions given in Loew's article. The filtrate of the lead precipitate, however, after freeing from lead, did not give any precipitation with alcohol in my case. Nor could a precipitation be obtained with FeCl 3 . 20 Therefore the ferment in my case did not resist precipitation by lead. The lead precipitate was now suspended in a small amount of water, the lead taken out by H 2 S, and the lead sulphide filtered off. The filtrate gave a very small precipitate with alcohol, which on dissolving in water showed no fermentative activity whatever. Hence this method was of no advantage for my purpose. The method used by Frankel und Hamburg 21 for purifying diastase, although based on good principles, is very tedious, and has not yet been tried with my ferment. Besides, I notice that the results of these authors could not be confirmed by a recent investigator. 22 PROPERTIES OF THE YEAST EXTRACT Besides having a characteristic action on glucose, the ferment extract has some other properties which deserve attention. In the first place let us consider its qualities as an oxydase. If a small portion of the extract or of the purified powder (alcohol purification) is added to a dilute solution of hydroquinone, and the mixture shaken, it turns red after a few minutes' standing. A similar action takes place with pyrogallol, the mixture turning yellow on short standing. No color change occurs with guajacol, 19 H. Euler, Allgemeine Chemie der Enzyme, p. 13, 1910. 20 See W. Lob und Pulvermacher, Biochem. Zeitschr., vol. 29, p. 316. 21 S. Frankel und M. Hamburg, Uber Diastase. 1: Versuche zur Her- stellung von Keindiatase und deren Eigenschaften, Hofmeister's Beitrdge zur chem. Pliysiol. u. Path., vol. 8, p. 389. 22 F. Miinter, tiber Enzyme 2te Mitt., Landw. Jahrbiicher, vol. 39, Erg. Bd. 3, p. 298, 1910; cf. Zentralbl. f. Biochemie und Biophysik., vol. 11, p. 185, 1910-1911. 1912 ] BircJcner: Yeast Glucase 175 nor with tincture of guajacum, not even after adding some hydrogen peroxyde. In this respect the ferment resembles Euler's Medicago laccase (loc. cit.}. As we have already seen before, 23 it also resembles Bertrand's Rims laccase (loc. cit.) in being accelerated in its action by manganese salts. The indophenol test (Rohmann-Spitzer's reagent) 24 was negative; the same w r as also the case with Tollens' 25 orcin reaction and Goldschmidt 's test. 26 Tollens' reaction with naphtoresorcin was also negative. No coloration occurred with a-napthol, nor w T ith tannic acid, the latter, however, giving a precipitate. Molisch's test 27 with a-naphtol, as well as Neuberg's pyrrol reaction, 28 was distinctly positive, even with the prepara- tion which had been precipitated with alcohol, and redissolved for three times. The carbohydrate group is therefore possibly in firm combination with the ferment molecule and may be regarded as one of its essential constituents. Possibly this factor is of significance for determining the specifity of the ferment (see Armstrong, loc. cit., p. 58). The ferment in aqueous solution is slightly dextrorotatory; it does not reduce Fehling's solution. It causes no color change on being added to a solution of tyrosin, and therefore contains no tyrosinase. It has a slight action on sodium lactate, liberat- ing an acid, without the formation of gas. The ferment gives all protein reactions ; boiling for one hour does not cause any pre- cipitation ; it is precipitated by alcohol and ether. The yeast extract contains evidently at least two different ferments : 23 See above, p. 170. 24 F. Kohmann und W. Spitzer, tiber Oxydationswirkungen tierischer Gewebe, Ber. Deutschr. diem. Ges., vol. 28, p. 567, 1895. 25 Loc. cit. 20 G. Goldschmidt, Eine neue Eeaction auf Glucuronsaure, Zeitschr. f. Physiol. Chemie, vol. 65, p. 390, 1910. 27 H. Molisch, Zwei neue Zuckerreaktionen, Monatschefte f. Chemie, vol. 7, p. 198, 1886. 28 C. Neuberg, tiber den Nachweis der Bernsteinsaure, Zeitschr. f. Physiol. Chemie, vol. 31, p. 574, 1900; Zur Kenntnis der Pyrrolreaktion, Chem. Centralbl., vol. 2, p. 1435, 1904. 176 University of California Publications in Physiology [VOL. 4 1. An oxydase, active against poly phenols, which is inactivated by boiling. 2. A glucolytic ferment which is not destroyed by heat, not even by boiling for fifteen minutes in a pressure flask. The latter ferment deserves particular attention. STUDIES ON THE PRODUCTS OF GLUCOSE FERMENTATION Numerous attempts have been made to obtain some knowledge about the substance into which glucose is transformed under the influence of this ferment. If glucose-ferment mixtures are filled into Schrotter's fer- mentation bulbs, and placed in the 70 incubator, the progress of the reaction can be observed very plainly. The liquid, if con- taining a fairly active extract, became distinctly acid within a few hours, and by and by the coloration took place. No gas formation was observed in any case. The iodoform reaction for alcohol was negative, but with Pasteur's droplet test, if applied in the manner as recently described by Klocker, 29 the presence of traces of alcohol, or of a similar substance, w r as ascertained. The sugar is therefore mainly transformed into acids. The access of air causes the reaction to proceed slightly faster, but it is not at all necessary, as I have convinced myself in special experiments. In tightly stoppered flasks, which were filled nearly to the top, leaving only about 1 c.c. of air space, the darkening and the formation of the dark residue occurred almost as readily as in other flasks that were provided only with cotton plugs. As a matter of course, toluol was added to all cultures, although the high temperature alone would nearly suppress bacterial action. I tried several ways of decolorizing the dark mixture with the object of rendering possible the application of the polariscope method. All these trials were unsuccessful so far. In the mean- while I have found, however, that a complete decolorization of the mixture is possible by means of a combined precipitation 20 A. Klocker, Nachweis kleiner Alkoholmengen in garenden Fliissig- keiten, Centralbl. f. Bacteriologie (n), vol. 31, p. 108, 1911. 1912] Birckner: Yeast Glucase 177 with mercuric acetate and phosphotungstic acid in the manner recently recommended by Neuberg. 30 All color reactions with this mixture naturally had to be unreliable, partly in direct consequence of the color, partly on account of the many substances present. Tollens' orcin reaction alone indicated with some certainty the presence of pentoses. The dark mixture, if added to Fehling's solution, reduced it rapidly in the cold. As according to Neuberg 31 only a few sugar derivatives show this behavior, namely glucuronic acid, glycerose, dioxyacetone, and glucose, I was led to believe, also, with regard to the recent results of Jolles, 32 that glucuronic acid was one of the products. As at that time I did not have at hand the reagents to make sure of this by a qualitative color reaction, I started a distillation process, following largely the 'suggestions given by C. Tollens. 33 The mixture, after filtering and cooling, was precipitated with basic lead acetate (no ammonia being added on account of the glucose), the precipitate washed with water, and boiled directly with HC1 (sp. g. 1.060) in the dis- tilling apparatus. Part of the filtrate from the lead precipitate was also distilled over with HC1, as this portion would contain any pentose that might possibly have been formed. If the distillate gave the furfurol test with aniline acetate (see C. Tollens, loc. cit.} the distillation was continued, until about 450 c.c. had been passed over. At the end, a solution of pure phloroglucin (Kahlbaum) in HC1 was added, and the amount of furfurol-phloroglucide estimated after sixteen hours' stand- ing. In no case did the lead precipitate, if properly washed, give even a trace of this substance, while the filtrate quite regularly gave a fair amount of phloroglucide. This filtrate, of course, still contained a large amount of unaltered glucose, and it seemed not absolutely certain that the furfurol was really 30 C. Neuberg und M. Ishida, Die Bestimmung der Zuckerarten in Naturstoffen, Biochem. Zeitschr., vol. 37, p. 142, 1911. si C. Neuberg, Die Physiologic der Pentosen und der Glucuronsaure, Ergebn. d. Physiol., vol. 3i, p. 387, 1904. 32 Biochem. Zeitschr., vol. 34, p. 242. ss C. Tollens, Quantitative Bestimmung der Glueuronsaure im Urin mit der Furfurol HC1. Destinations methode, Zeitschr. f. Physiol. Chemie, vol. 61, p. 95, 1910. 178 University of California Publications in Physiology [VOL. 4 derived from pentose. 34 To investigate this point, I have in one case treated the filtrate of the lead precipitate with H 2 S, thus removing all the lead. The H 2 S was driven out of the dark yellowish filtrate by a current of air, and the liquid fermented with yeast. The solution soon turned dark crimson, and, although perfectly clear, assumed a strong odor, similar almost to that of indol and skatol. The tests for these substances, how- ever, gave negative results, as was to be expected. At the end of the fermentation the reducing power of the liquid against Fehling's solution was strongly diminished, and the Cu 2 O formed had a peculiar crimson color. That the reducing agent in this case was not glucose was ascertained by preparing the osazone. A sample of the liquid was boiled in the water bath for one hour with phenylhydrazine hydrochlorate and sodium acetate. It was then allowed to cool very gradually. On exam- ining the substance under the microscope after several hours, I observed only traces of glucosazone crystals, while the liquid was filled with masses of small brownish globule-like crystals of an oily appearance. This is exactly the manner in which the arabinosazone is described by von Lippmann 35 to appear when first formed in the presence of foreign bodies. In fact, 011 prolonged standing, a solid yellowish-brown sediment of osazone crystals separated out. On examining, the long, needle-shaped crystals of a light yellow color were easily seen although they were densely covered with a rust-brown, amorphous substance. On distilling the liqui' over with HC1, and adding phloroglucin to the distillate, an r uple precipitation took place. Hence the formation of pentose seemed sufficiently assured; while any for- mation of glucuronic acid had to be denied. Quantitative studies of this pentose formation are in progress. "With one of the early digests, which contained 40 per cent glucose, I made an ether extraction, shaking the mixture with ether in a separating funnel, and using fresh ether several times. The first portions of this extraction were unfortunately lost. 3-11 00 g. glucose may yield on distillation with HC1 up to 0.222 g. furfurol (Stoklasa; cf. v. Lippmann, loc. cit., p. 103). <" von Lippmann, Die Chemie der Zuckerarten (Braunschweig, 1904), p. 91. 1912] Birckner: Yeast Glucase 179 With the dark oily syrup at the bottom and ether on top, the funnel was put aside for a long time. Finally, on testing, I found that the ether had become strongly acid. Still this acid apparently was not very soluble in ether, as it had not been removed by the first three or four portions of ether. Besides, when I tried to obtain a similar extraction from an ether digest, which did not contain such a high concentration of glucose, the acid, for the most part, stayed in the aqueous solution, and the ether became only very faintly acid. The strongly acid ether fraction, on evaporating, yielded a yellowish, oily fluid of a peculiar odor. This liquid was taken up in water and carefully distilled. The distillate was a color- less, neutral fluid of aldehydic odor, which did not give pre- cipitates with CaCl,, FeCl 3 , or alcohol. It gave, however, a fine bluish-white precipitate with AgN0 3 , which turned greyish brown on heating. The tests for aldehyde gave negative results. This substance was not identified. The acid residue, in the distilling flask, did not give any pre- cipitate except with lead salts. It was optically inactive and did not reduce Fehling's solution. Tollens' orcin reaction, as well as the napthoresorcin reaction and Goldschmidt 's test (loc. cit.) was negative. Molisch's test with a-naphtol was positive. On evaporating in the vacuum over sulphuric acid, the acid liquid did not crystallize but yielded a dark syrup. If to this syrup a concentrated solution of potassium acetate was added, crystals were formed almost immediately. These granular crystals could also be obtained a dilute solution in a test tube, if after adding potassium acetate the walls of the test tube were rubbed with a glass rod. I obtained just enough of these crystals to make a few melt- ing point determinations. The substance did not melt until above 300 C. I prepared the acid potassium salts of saccharic acid and of tartaric acid in order to compare their respective melting points with the one obtained. The melting point of the saccharate was found to be 186?5 C., that of the tartrate about 270 C., while the melting point of the acid potassium oxalate, which salt is rather insoluble, too, was found to lie above 350 C. I presume that the crystals which I had obtained on addition 180 University of California Publications in Physiology .4 of CH 3 COOK, were the result of some sort of secondary trans- formation of the original acid into oxalic acid. Primarily, the ether soluble extract did certainly not contain oxalic acid, as no crystals were obtained on evaporation and as the calcium pre- cipitate was readily soluble in acetic acid (see table below). If the free acid was just neutralized with ammonia, several characteristic reactions could be obtained, the results of which I have arranged in the following table : Free acid Reagent No precipitate AgN0 3 No precipitate FeCl 3 No precipitate CaCl 2 No precipitate Alcohol Ammonia salt Yellow precipitate; soluble in the cold in NH 3 . On heating, slight reduction. White, gelatinous precipitate, which dis- solves slowly in N/10 HC1, more rapidly in strong HC1. Also dissolves in excess of NH 3 to a dark yellow solution (of FeCl 3 color). Thick white precipitate. Insoluble when heated, and in cold NaOH. Dissolves in N/10 HC1, and also on adding a few drops of glacial acetic acid. ll'Iiilc precipitate. Insoluble in ether. On evaporating the solution of the ammonia salt, a greyish, crystalline mass of caramel-like odor was obtained. Under the microscope it appeared to consist largely of rhombic plates. No exact melting point could be obtained with this residue. Prob- ably it was not a single compound, but a mixture of several salts'. Above 180 C. the substance in the capillary tube turned brownish, but it had not melted yet at a temperature of 250 C. A portion of the dark residue of the ether extraction which I mentioned above, was diluted with water and carefully dis- tilled; the receiver being cooled by ice-water. The colorless clear distillate had a strong odor and was distinctly acid against litmus paper. It gave the following reactions : (a) It reduced ammoniakal silver solution. (6) After acidifying a small portion with H 2 SO 4 , it was shaken in a separating funnel with pure chloroform. The chloroform was drawn off; and on adding to it 0.5 c.c. of Nessler's reagent (freshly prepared), a strong yellow coloration of the latter occurred at once. 1912] Birckner: Yeast Glucase 181 (c) A crystal of resorcin was dissolved in a few drops of the liquid, and the mixture allowed to run down slowly along the side of a test tube containing concentrated sulphuric acid. At the zone of contact a bright, orange red color developed very soon, which gradually turned darker red. (d) The distillate gave Schryver's 30 formaldehyde test very plainly. The reaction was carried out in the follow- ing way : To 5 c.c. of the liquid were added 1 c.c. of a 1 per cent solution of phenylhydrazine chlorhydrate (freshly prepared and filtered), 0.5 c.c. of a freshly prepared 5 per cent solution of potassium ferri-cyanide, and 2.5 c.c. of strong HC1 (sp. g. 1.19). The mixture showed a distinct, though not very intense red coloration. It was now shaken with pure ether in a small separating funnel, and the lower layer drawn off. To the ethereal extraction were now added 1-2 c.c. of strong HC1. The latter assumed almost immediately a very intense red color, indicating the the presence of formaldehyde. In addition to this substance, the distillate, as already men- tioned, contained a volatile acid (possibly formic acid), which however, I have not yet identified. In view of the fact that W. Lob (loc. cit.) has found as the products of electrolysis of glucose polyoxyacids, pentose, and formaldehyde, it is certainly of some interest that, as I have shown, a ferment-like substance occurs in the yeast cell which is able to bring about (or rather to accelerate) the cleavage of the glucose molecule into essentially the same products. 3c S. B. Schryver, The photochemical formation of formaldehyde in green plants, Eoy. Soc. Proc., 82, p. 226, 1910. 182 University of California Publications in Physiology [VOL. 4 CONCLUSIONS Summarizing briefly the contents of this article, I may state the following: 1. A systematic review has been given in the first part of the paper of some recent advances of our knowledge of glucose oxydations and cleavages, both outside and inside of the organism. 2. In the second part of this article a ferment has been described which occurs in the California steam-beer yeast under certain conditions, and which has the property of accelerating the decomposition of glucose at an elevated temperature. 3. This new ferment is not identical w r ith zymase. It acts preferably at a temperature of 70 C. It causes no gas forma- tion and yields no alcohol. 4. Its action on glucose at 70 C. manifests itself by a rapid darkening of the mixture, an increase in acidity, a gradual formation of a carbon-like solid settlement, and the development of an odor similar to that of caramel. 5. The ferment may be extracted from a yeast powder (Dauerhefe) -which is best obtained by killing the cells with ethyl alcohol. 6. From a w r atery extract the yeast glucase may be obtained and purified by repeated precipitation with alcohol; but this process always involves a weakening of the ferment. 7. Yeast glucase is very stable in aqueous solution, if kept at room temperature under sterile conditions. Boiling does not destroy its activity. 8. The yeast glucase preparation shows activity in neutral or acid solution against glucose, polyphenols, and lactates. It does not contain tyrosinase, nor does it act as a peroxydase against, glucose. 9. The ferment preparation gives a strong pyrrol reaction (Neuberg). 10. Yeast glucase shows some relationship to the oxydases, but with regard to its main function, it is to be classed together with zymase in a group which stands separately from the Birckner: Yeast Glucase 183 oxydases and the hydrolytic ferments, and to which Euler has applied the name " Garungsenzyme " (see p. 154). 11. The transformation products of glucose resulting from the action of this ferment are partly acids, none of which has so far been definitely identified. However, among the cleavage products of the sugar the presence of pentose and of formalde- hyde could be ascertained. I wish to express my thanks to Dr. T. Brailsford Robertson for his valuable counsel and continuous interest in this in- vestigation, and also to Mr. C. B. Bennett for kind suggestions in carrying out some of the chemical work. Transmitted March 9, 1912. ; UNIVEBSITY OF CALIFORNIA PUBLICATIONS (Continued) 21. On the Local Application of Solutions of Saline Purgatives to the Peritoneal Surfaces of the Intestines, by John Bruce MacCallum. Pp. 187-197. July, 1904. Nos. 20 and 21 in one cover .25 22. On the Toxicity of Distilled Water for the Fresh-water Gammarus. Suppression of this Toxicity by the Addition of small quantities of Sodium Chloride, by G. Bullot. Pp. 199-217. July, 1904 .20 Vol.2. 1. The Control of Heliotropic Reactions in Fresh-water Crustaceans by Chemicals, especially CO 3 (a preliminary communication), by Jacques Loeb. Pp. 1-3. November, 1904 05 2. Further Experiments on Heterogeneous Hybridization in Echinodenns, by Jacques Loeb. Pp. 5-30. December, 1904 8. Influence of Calcium and Barium on the Secretory Activity of the Kidneys (second communication), by John Bruce MacCallum. Pp. 31-42. December, 1904. 4. Note on the Galvanotropic Reactions of the Medusa Poly orchis penicillata A. Agassiz, by Frank W. Bancroft. Pp. 43-46. Decem- ber, 1904. Nos. 2, 3 and 4 in one cover .45 6. The Action on the Intestines of Solutions containing two Salts, by John Bruce MacCallum. Pp. 47-64. January, 1905. 6. The Action of Purgatives in a Crustacean (Sida crystallina), by John Bruce MacCallum. Pp. 65-70. January, 1905. Nos. 5 and 6 in one cover .25 7. On the Validity of Pfluger's Law for the Galvanic Action of Para- mecium . (preliminary communication), by Frank W. Bancroft. P. 71. February, 1905. 8. On Fertilization, Artificial Parthenogenesis and * Cytolysis of the Sea-urchin Egg, by Jacques Loeb. Pp. 73-81. February, 1905. Nos. 7 and 8 in one cover , J.5 9. On an Improved Method of Artificial Parthenogenesis, by Jacques Loeb. Pp. 83-86. February, 1905 .05 10. On the Diuretic Action of Certain Haemolytics, and the Action of Calcium in Suppressing Haemoglobinuria (preliminary communica- tion), by John Bruce MacCallum. Pp. 87-88. March, 1905. 11. On an Improved Method of Artificial Parthenogenesis (second com- munication), by Jacques Loeb. Pp. 89-92. March, 1905. Nos. 10 and 11 in one cover 05 12. The Diuretic Action of Certain Haemolytics and the Influence of Calcium and Magnesium in Suppressing the Haemolysis (second communication), by John Bruce MacCallum. Pp. 93-103. May, 1905. 13. The Action of Pilocarpine and Atropin on the Flow of Urine, by John Bruce MacCallum. Pp. 105-112. May, 1905. Nos. 12 and 13 in one cover 25 14. On an Improved Method of Artificial Parthenogenesis (third com- munication), by Jacques Loeb. Pp. 113-123. May, 1905 15 15. On the Influence of Temperature upon Cardiac Contractions and its Relation to Influence of Temperature upon Chemical Reaction Velocity, by Charles D. Snyder. Pp. 125-146. September, 1905 .25 16. Artificial Membrane Formation and Chemical Fertilization in a Star- fish (Asterina), by Jacques Loeb. Pp. 147-158. September, 1905 15 17. On the Influence of Electrolytes upon the Toxicity of Alkaloids (pre- liminary communication), by T. Brailsford Robertson. Pp. 159-162. October, 1905 05 18. Studies on the Toxicity of Sea-water for Fresh-water Animals (Gammarus pulex De Geer), by C. H. Wolfgang Ostwald. Pp. 163-191; plates 1-6. November, 1905 .35 19. On the Validity of Pfliiger's Law for the Galvanotropic Reactions of Paramecium* by Frank W. Bancroft. Pp. 193-215; 8 text figures. November, 1905 . .20 Vol.3. 1. On Chemical Methods by which the Eggs of a Mollusc (Lottia Gigantea) can be caused to become Mature, by Jacques Loeb. Pp. 1-8. November, 1905 05 2. On the Changes in the Nerve and Muscle which seem to Underlie the Electrotonic Effect of the Galvanic Current, by Jacques Loeb. Pp. 9-15. December, 1905 05 3. Can the Cerebral Cortex be Stimulated Chemically? . (Preliminary" communication), by S. S. Maxwell. Pp. 17-19. February, 1906.. .05 4. The Control of Galvanotropism in Paramecium by Chemical Sub- stances, by Frank W. Bancroft. Pp. 21-23. March, 1906 .10 5. The Toxicity of Atmospheric Oxygen for the Eggs of the Sea-urchin (Strsngyloeentrotus purpuratus) after the Process of Membrane Formation, by Jacques Loeb. Pp. 33-37. March, 1906. UNTVEESITY OF CALIFORNIA PUBLICATIONS (Continued) 6. On the Necessity of the Presence of Free Oxygen in the Hypertonic Sea-water for the Production of Artificial Parthenogenesis, by Jacques Loeb. Pp. 39-47. March, 1906. Nos. 5 and 6 in one cover 15 7. On the Counteraction of the Toxic Effect of Hypertonic Solutions upon the Fertilized and Unfertilized Egg of the Sea-urchin by Lack of Oxygen, by Jacques Loeb. Pp. 49-56. April, 1906... .05 8. On the Production of a Fertilization Membrane in the Egg of the Sea-urchin with the Blood of Certain Gephyrean Worms (a pre- liminary note), by Jacques Loeb. Pp. 57-58. March, 1907 . .05 9. Note on the Synthesis of a Protein through the Action of Pepsin (preliminary communication), by T. Brailsford Robertson. Pp. 59-60. April, 1907 ;..... .05 10. The Chemical Character of the Process of Fertilization, and its Bear- ing upon the Theory of Life-Phenomena, by Jacques Loeb. Pp. 61-80. September, 1907 25 11. A New Proof of the Permeability of Cells for Salts or Ions (a pre- liminary communication), by Jacques Loeb. Pp. 81-86. January, 1908 .05 12. The Origin of two new Retrogressive Varieties by one Mutation in Mice, by Arend L. Hagedocrn. Pp. 87-90. September, 1908 05 IS. On Synthesis of Paranuclein through the Agency of Pepsin and Chemi- cal Mechanics of Hydrolysis and Synthesis of Proteins through the Agency of Enzymes, by T. B. Eobertson. Pp. 91-94. December, 1908 05 14. The Inheritance of Yellow Color in Eodents, by Arend L. Hagedoorn. Pp. 95-99. March, 1909 05 15. Table of H + and OH~ Concentrations corresponding to Electromotive Forces determined in Gas-chain measurements, by C. L. A. Schmidt. Pp. 101-113. September, 1909 10 16. The Proteins, by T. Brailsford Eobertson. Pp. 115-194. October, 1910 $1.00 17. Further Proof of the Identity of Heliotropism in Animals and Plants, by Jacques Loeb and S. S. Maxwell. Pp. 195-197. January, 1910 .05 Vol.4. 1. Experiments on the Function of the Internal Ear, by S. S. Maxwell. Pp. 1-4. September, 1910 05 2. On the Eise of Temperature in Eabbits, Caused by the Injection of Salt Solutions, by Theo. C. Burnett. Pp. 5-7. September, 1910 05 3. A Biochemical Conception of Dominance, by A. E. Moore. Pp. 9-15. September, 1910 05 4. Galvanotropic Orientation in Gonium pectorale, by A. E. Moore and T. H. Goodspeed. Pp. 17-23. May, 1911 05 5. On a Possible Source of the Biological Individuality of the Tissues and Tissue-fluids of Animal Species, by T. Brailsford Eobertson. Pp. 25-30. May, 1911 05 6. Some Factors Influencing the Quantitative Determination of Gliadin, by J. E. Greaves. Pp. 31-74. August, 1911 40 7. Errors of Eefraction Occurring in the Students of the University of California, by Theo. C. Burnett. Pp. 75-77. August, 1911 05 8. On the Cytolytic Action of Ox-Blood Serum upon Sea-Urchin Eggs, and Its Inhibition by Proteins (Preliminary communication), by T. Brailsford Eobertson. Pp. 79-88. February, 1912 10 9. On the Nature of the Cortical Layer in Sea Urchin Eggs, by A. E. Moore. Pp. 89-90. March, 1912. 10. On the Nature of the Sensitization of Sea Urchin Eggs by Strontium Chloride, by A. E. Moore. Pp. 91-93. March, 1912. Nos. 9 and 10 in one cover .05 11. On the Isolation of Ob'cytase, the Fertilizing and Cytolyzing Substance in Mammalian Blood Sera, by T. Brailsford Eobertson. Pp. 95-102. March, 1912. 12. On the Extraction of a Substance from the Sperm of a Sea Urchin (Strongylocentrotus purpuratus) which will Fertilize the Eggs of that Species, by T. Brailsford Eobertson. Pp. 103-105. March, 1912. 13. The Demonstration of "Masked" Iron in Blood, by C. B. Bennett. Pp. 107-108. March, 1912. Nos. 11, 12 and 13 in one cover 10 14. A New Method of Heterogeneous Hybridization in Echinoderms, by A. E. Moore. Pp. 109-110. March, 1912. 15. Can the Presence of Acid Account for the Oedema of Living Muscle, by A. E. Moore. Pp. 111-114. April, 1912. Nos. 14 and 15 in one cover ..: 05 16. On the Oxydations and Cleavages of Glucose. Yeast Glucase, a New Glucolytic Ferment, by Victor Birckner. Pp. 115-183. September, 1912 75 Other series: American Archaeology and Ethnology, Botany, Classical Philology, Eco- nomics, Engineering, Entomology, Geology, History, Lick Observatory Bulletins, Lick Ob- servatory Publications, Mathematics, Modern Philology, Pathology, Philosophy, Psychology* Publications of the Academy of Pacific Coast History, and Zoology. NON-CIRCUUTING BOOK