ADVANCES IN ENZYMOLOGY
`
`AND RELATED SUBJECTS OF
`BIOCHEMISTRY
`
`Volume XI
`
`Eton Ex. 1059
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`
`

`

`CONTRIBUTORS TO VOLUME XI
`
`E. S. GUZMAN BARRON, Chemical Division, Department of Medicine,
`The University of Chicago, Chicago, Illinois
`J. F. DANIELLI, Department of Zoology, King’s Collage, London
`W. C. 8, England
`J. T . DAVIES, Department of Chemistry, King’s College, London
`W. C. 8, England
`ERNST GAUMANN, Institut f u r spezielle Botanik der Eidgenassischen
`Technischen Hochschule, Zurich, Switzerland
`HERBERT GUTFREUND, Department of Colloid Science, University of
`Cambridge, Cambridge, England
`EDWARD J. HEHRE, Department of Bacteriology and Immunology,
`Cornell University Medical College, New York
`EUGENE F. JANSEN, Enzyme Research Division, Western Regional
`Research Laboratory, United States Department of Agriculture,
`Albany 6, California
`HANS LINEWEAVER, Western Regional Research Laboratory, United
`States Department of Agriculture, Albany 6, California
`STANLEY PEAT, Chemistry Department, Unicersity College of North
`Wales, Bangor, Caernarvonshire, Wales
`EWALD SEEBECR, Sandoz Ltd., Basel I S , Switzerland
`ARTHUR STOLL, Sandoz Ltd., Basel IS, Switzerland
`E. C. WASSINK, Laboratory for Plant Physiological Research, Agri-
`cultural University, Wageningen, Netherlands
`
`Eton Ex. 1059
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`

`

`ADVANCES IN ENZYMOLOGY
`AND RELATED SUBJECTS OF BIOCHEMISTRY
`
`E d i t e d b y F . F . N O R D
`
`FORDHAM UNIVERSITY, N E W YORK, N. Y .
`
`VOLUME XI
`
`N E W Y O R K
`INTERSCIENCE PUBLISHERS LTD., LONDON
`
`Eton Ex. 1059
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`
`

`

`Copyright, 1951, by
`
`I N T E R S C I E N C E P U B L I S H E R S , INC.
`
`All Rights Reserved
`
`This book or any part thereof must not
`be reproduced without permission of the
`publisher in writing. This applies spe-
`cifically to photostat and microfilm
`reproductions.
`
`IN'I'E KS C1 E N C E P U B LI S H E R S , I N C .
`250 Fifth Avenue, New York 1, N. Y.
`For Great Britain and Northern Ireland:
`
`I N T E R S C I E N C E P U B L I S H E R S , LTD.
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`
`Printed in the United States of America
`by Mack Printing Company, Easton, Pa.
`
`Eton Ex. 1059
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`

`

`C O N T E N T S
`The Nature of Entropy and Its Role in Biochemical Processes . By
`HERBERT GUTFREUND. Cambridge. England .....................
`I . The Nature of Entropy ........................................
`A . 1ntroduct.ion .............................................
`B . Historical . . . . . . . . . . . . .
`......................
`C . Some Fundamental Defi
`............................
`D . Entropy of Svstems in Equilibrium .........................
`E . Entropi of Irreversible Piocesses and Open Systems ..........
`F . Procedures for Experimental Determination of Entropy Changes
`I1 . Entropy Changes in Some Selected Processes .....................
`A . Ehtropy and Change of State of a Gas ......................
`B . Entropy of Mixing: Osmotic and Diffusion Processes .........
`C . Entropy and Elasticity of Fibers ...........................
`D . Entropy and Chemical Equilibria and Reactions .............
`I11 . Conclusions about the Nature and Role of Entropy ...............
`....................................
`References ...........
`Reactions at Interfaces in Relation to Biological Pro
`............
`DANIELLI and J . T . DAVIES, London, England ....
`I . Introduction ..................................................
`I1 . Distribution of Soluble Ions a t Interfaces ........................
`TI1 . Partition of SH Groups between Surface and Bulk Phases ..........
`A . Nonionogenic Thiols ......................................
`B . Ionogenic Thiols .........................................
`C . Effect of Variation in Ionic Strength ............
`D . Effect of Variation in Bulk Thiol Concentration ..............
`E . Significance for Studies with Enzymes ......................
`F . Redox Indicators ...............................
`G . Other Surface SH Problems ...............................
`IV . Partition of Reactants between Surface and Bulk Phases .
`V . Factors Influencing Rate of an Interfacial Reaction . . . . .
`A . Pressure, Temperature, and Tightness of Packing of Molecules
`or Ions in the Interface .................................
`B . Stereochemical Configuration of Reactant Molecules ..........
`C . Changed Ionic Concentrations a t the Interface ...............
`D . Rates of Diffusion to and from the Interface of Reactants and
`Products, Respectively .................................
`VI . Oxidation and Reduction Phenomena in an Interface ..............
`A . Toxicity of Ions ..........................................
`B . Effect of Position of Double Bonds in Sterols on Their Oxidation
`C . Photoxidation and Surface Potential ........................
`VII . Some Particular Surface Reactions ..............................
`A . Digestion of Esters by Pancreatin ..........................
`B . Action of Snake Venoms on Surface Films ...................
`C . Photochemical Reactions in Monolayers ....................
`VIII . Interactions and Complex Formation in Monolayers ...............
`IX . Reactions Involving Two Surface Phases .........................
`X . General Discussion ............................................
`References ...................................................
`V
`
`~~
`
`2
`3
`4
`7
`7
`10
`10
`14
`14
`14
`14
`15
`15
`23
`26
`30
`32
`
`35
`35
`37
`47
`48
`49
`51
`53
`54
`55
`55
`56
`59
`
`59
`64
`67
`
`67
`69
`69
`74
`76
`76
`76
`77
`79 ..
`82
`85
`86
`87
`
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`

`

`vi
`
`CONTENTS
`
`Chlorophyll Fluorescence and Photosynthesis. By E. C. WASSINK,
`Wagenzngen, Netherlands.. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`I. Introduction. . . . . . . . , , . . . . . . ,
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`11. The Work of Kautsky et al.. . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . .
`111, Studies of the Utrecht-Dclft Gr
`IV. The Work of McAlwter and Myers.. . . . . . . . . . . . . . . . . . . . . . . . . .
`. .
`V. The Investigations of Franck et al.. . . . . . . . . . . . . . . . . . . . . . . . . .
`r Veen and Others.. . . . . . . . . . . . . . . . . . .
`VI. Observation
`VII. Conclusions
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`References.
`
`91
`92
`94
`119
`150
`158
`189
`195
`197
`
`201
`202
`
`Thiol Groups of Biological Importance. By E. S. GUZMAN BARRON,
`Chicago, Illinms. . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . - . . . . - . . . . - .
`A. Oxidation by Oxygen., , . , . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . .
`B. Other oxidizing Agents.. . . . . . , , . . . . . , . . . . . . . . . . . . . . . . . , . .
`C. Photochemical Oxidation-Reduction . , . . . . . . . . . . . . . . . . . . . . . .
`D. Oxidation-Reduction Potentials, . . . , . . . . . . . . . . . . . . . . . . . . . . .
`E. Alkylating
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`F. Mercaptid
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`f Biological Significance. . . . . . . . . . . . . . . . . . .
`G. Other Rea
`111. Thiol Groups in Proteins.. . . . . . . . . . . . . . . . . .
`. . . . . . . . , . .
`A. Types of -SH Groups.. . . . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . .
`B. Denaturing Agents.. . . . . . . . . . . . . . . . . . . .
`C. Oxidizing Agents.. . . . . . , , . . . , . , , . . . . . . . . . . . . . . . . . . . . . . . . .
`1. Ferricyanide.. . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2. Porphyrindin. . . . . . . . . . . . . . . . . . . . .
`3. Iodosobenzoate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`gents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`D. Alkylating Agents.. . . . . . . . . . . . . . . . . , , . . . . . . . . . . . . . . . . . . . .
`E. Mercaptide-Forming Agents. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`F. Reducing Agents.. . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . .
`. . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`V. T~iolEnzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Thiol Groups Essential in Enzyme Activity. . . . . . . . . . . . . . . .
`B. Thiol Reagents for Enzyme Activity.. . . . . . . . . . . . . . . . . . . . . .
`1. Oxid%ing Agents.
`. . . . . . . . . . . . . . . . . . . . . . . . . . .
`ts . . . . . . . . . . . . . . . . . . . . . . . . . .
`2. Mercaptide-Formi
`3. Alkylating Agents
`C. Reversal of Inhibition.
`D. Thiol Enzymes. . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`VI. Other Functions of Thiol Groups in Proteins. . . . . . . . . . . . . . . . . . . . .
`A. Toxins . . . . . . . . . . . . . . . . . . . . . , , , . . . . , , . . . . . . . . . . . . .
`. . .
`B. Coagulation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`C. Antibiotics. . . . , , . . . . . . . . . . . , , . . . . . , , . . . .
`246
`D. Permeability. . . . . . . . . . . . . . . . . . . . . . . . . . . .
`247
`E. Thiols and Hormones.. . . . . . . , , . . . . , , . . . . . . . . . . . . . . . . . . . . .
`247
`VII. Glutathione.. .
`. . . . . . . . . . . . . . . . . . . . .
`248
`A. Distributi
`. _ . . . . . . . . . . _ _ . _ . . _ . .
`248
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. Metabolis
`249
`C. Glutathione
`250
`. . . . . . . . . . . . . . . . . . . . .
`D. Glutathione and Enzymic Reactions. . , . . . . . . . .
`250
`1. Protection
`250
`252
`2. Regulatory Function
`. . . . . . . . . . . . . . . . . . . . . . .
`E. Thiols and Ox gen Tension.. . . . , . . . . , . . . . . . . . . . . . . . . . . . . . .
`252
`F. Thiols and Ce8 Division and Growth.. , . . . . . . . . . . . . . . . . . . . .
`253
`G. Thiols and Ionizing Radiations. . . . . . . . . .
`255
`
`211
`213
`216
`218
`219
`219
`221
`223
`223
`
`225
`226
`227
`228
`229
`232
`233
`235
`
`240
`
`Eton Ex. 1059
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`
`

`

`CONTENTS
`
`Thiol Groups of Biological Importance (continued)
`VIII. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Pectic Enzymes. By HANS LINEWEAVER and EUGENE F. JANSEN,
`Albany, California.
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`11. Nomenclature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`111. Enzymic Hydrolysis of Pectic Substances. . . . . . . . . . . . . . . . . . . . . . . .
`A. Mode of Pectinesterase Action.. . . . . . . . . . . . . . . . . . . . . . . . .
`B. Mode of Polygalacturonase Action.. . . . . . . . . . . . . . . . . . . . . . . .
`IV. Pectinesterase (General Characteristics). . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`D. Specificity. .......
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`E. Effect of Cations on Activity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`F. Preparation and Purification of Higher Plant Pectinesterase . .
`G. Miscellaneous Properties. .................................
`V. Polygalacturonase (General Characteristics). . . . . . . . . . . . . . . . . . . . . .
`A. Occurrence.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`C . Specificity ......
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`D. Purification of F
`1 Polygalacturonase
`E. Miscellaneous Properties. . . . . . . . . . . . . .
`VI. Pectic Enzymea Other Than Pectinesterase an
`VII. Use and Control of Pectic Enzymes., . . . . . . .
`VIII. Production of Pectic E
`References. . . . . . . . . . .
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`es: A Biological Type of Polymeriza-
`New York, N . Y.. . .
`. . . . .
`I. Introduction.
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`11. Formation of
`rides of thc Starch-Glycogen Class from Glu-
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. Phosphorylase plus Branching Factor., . . . . . . . . . . . . . . . . . . . . .
`111. Formation of Serologically React,ive Dextrans and Levans from Sucrose
`A. Dextransucrase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. Levansucrase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`IV. Formation of Polysaccharides of the Starch-Glycogen Class from SU-
`crose, Maltose, and Cycloamylose. . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Amylosucrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. Amylomaltase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`C. BaciUus mcerans Amylase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`V. Formation of Branched Amylopolysaccharides and Dextrans from
`Amylose-Type Chains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Q-Enzyme.. ...
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`B. Dextran-Dextrin
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`VI. General Discussion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`By STANLEY PEAT, Bangor,
`The Biological Transformations of Starch.
`Wales. . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . .
`. .
`I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Amylose and Amylopectin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . .
`11. Starch and Photosynthesis
`
`vii
`
`257
`258
`
`267
`
`27 1
`273
`277
`
`280
`
`286
`286
`287
`288
`290
`292
`293
`
`297
`297
`
`299
`299
`304
`306
`308
`312
`
`322
`
`329
`332
`
`339
`339
`339
`34 1
`
`Eton Ex. 1059
`7 of 74
`
`

`

`viii
`
`CONTENTS
`
`The Biological Transformations of Starch (continued)
`I11 . The Enzymes Involved in Starch Metabolism .....................
`A . Amylolysis ..............................................
`1 . Saccharogenic Amylases ..............................
`2 . Dextrinogenic Amylases ..............................
`B . Biological Synthesis of Starch ..............................
`1 . Character of Synthetic Starch ..
`2 . Mechanism of Synthesis ........
`C . Phosphorolysis ...................................
`D . The Branching Enzyme .........
`..............
`1 . Q-Enzyme ..........................................
`2 . The Phosphate Question ..............................
`3 . The Question of Reversibility ..
`. . . . . . . . . . . . . . . .
`4 . Energy Requirements ................................
`E . Starch and Sucrose ................
`References ...................................................
`Chemical Investigations on Alliin, the Specific Principle of Garlic .
`By
`ARTHUR STOLL and EWALD SEEBECK, Basel, Switzerland ........
`I . Historical Introduction ........................................
`I1 . Search for the Active Principle ..................................
`I11 . Outline of Method of Isolation of Alliiri ..........................
`IV . Properties of Alliin ............................................
`V . Composition and Constitution of Alliin . .
`.....................
`VI . Synthesis of Alliin and Its Three Isomers .......................
`VII . Alliinase .....................................................
`A . Enzymic Degradation of Alliin .............................
`B . Preparation and Properties of Alliinase Preparations ..........
`C . Specificity of Alliinase with Regard to Structure .............
`D . Specificity of Alliinase with Regard to Configuration ..........
`VIII . Questions Regarding the Therapeutic Use of Alliin ................
`References ...................................................
`Some Problems of Pathological Wilting in Plants .
`By ERNST GXUMANN, Zurich, Switzerland .......................
`I . Introduction ..................................................
`I1 . The Wilting Toxins ...........................................
`A . Chemical Nature of the Wilting Toxins in Plants .............
`B . Effect of the Wilting Toxins on the Host Plant ..............
`C . The Minimal Dose .......................................
`D . The Lyeomarasmine-Iron Complex .........................
`E . Effect of the Glucosans-Mechanical
`I11 . Host-Parasite Relationships . . . . . . . . . . . . . . . . . .
`. . . . . . . . . . . . . .
`A . Problem of Host Specificity and Tissu
`B . Toxigenic and Pathogenic Properties of the Parasite ..........
`C . Resistance of Host to Parasite and to Its Toxin ..............
`D . Influence of Nutrition on Sensitivity of Tomato Plants to the
`Wilting Toxin .........................................
`IV . Summary ....................................................
`....................................
`Bibliography ..........
`Author Index ......................................................
`Subject Index ......................................................
`..........................
`Cumulative Indexes . .
`. . .
`
`343
`344
`344
`347
`348
`352
`353
`3.59 ...
`361
`362
`366
`367
`369
`370
`373
`
`377
`377
`...
`378
`381
`383
`383
`387
`
`395
`397
`399
`
`401
`401 ...
`402
`402
`403
`410
`413
`416
`_ _ .
`422
`422
`426
`429
`
`432
`434
`435
`
`439
`
`453
`
`463
`
`Eton Ex. 1059
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`
`

`

`Advances in Enzymology and Related Areas of Molecular Biology, Volume 11
`Edited by F. F. Nord
`Copyright © 1951 by Interscience Publishers, Inc.
`
`THIOL GROUPS O F BIOLOGICAL
`IMPORTANCE*
`
`By E. S. GUZMAN BARRON, Chicago, Illinois
`
`C O N T E N T S
`
`202
`203
`206
`206
`210
`211
`213
`216
`218
`219
`219
`22 1
`223
`223
`224
`225
`225
`225
`226
`
`235
`
`I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`11. Some
`I1 . Some Pro erties of Thiols .....................................
`..................................
`A.
`A . Oxi&tion by Oxygen ....................................
`.........
`B . Other Oxidizing Agents ..................................
`B.
`C . Photochemical Oxidation-Reduction .......................
`.......................
`C.
`D . Oxidation-Reduction Potentials ...........................
`D.
`E . Alkylating Agents .......................................
`E.
`F.
`F . Mercaatides ............................................
`G. Other Reactions of Biological Significance. . . . . . . . . . . . . . . . . .
`111. Thiol Groups in Proteins. .....................................
`A. Types of -SH Groups.. . .
`B. Denaturing Agents. . .
`C. Oxidizing Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`1. Ferricyanide . . . . . . . .
`2. Porphyrindin ......................................
`3. Iodosobenzoate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`4. Iodine ....................
`5. Other Oxidizing Agents.. . . .
`..............................
`D. Alkylating Agents.. . . . . . . . . . . . .
`E. Mercaptide-Forming Agents. . . . .
`F. Reducing Agents. . . . . . . . . . . . . . .
`IV. Myosin.. ......
`.....................
`. . . . . . . . . .
`V. Thiol Enzymes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Thiol Groups Essential in Enzyme Activity.. . . . . . . . . . . . . . . .
`B. Thiol Reagents for Enzy
`1. Oxidizing Agents. .
`. . . . . . . . . . . . . . . . . . . . . . . . . .
`2. Mercaptide-Formin
`3. Alkylating Agents.
`C. Reversal of Inhibition.. .
`239
`
`.
`.
`.
`.
`.
`D. Thiol Enzymes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`240
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y7"
`VI. Other Functions of Thiol Groups in Proteins.. . . , . . . . . . . . . . . . . . . .
`244
`B. Coagulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`A. Toxins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`244
`C. Antibiotics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`246
`D. Permeability. ,
`247
`E. Thiols and Hormones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`247
`VII. Glutathione. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`248
`A. Distribution. .
`248
`B. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`249
`C. Glutathione and Ascorbic Acid.. ..........................
`250
`
`U . -111".
`
`" " " J A . ' U U . .
`
`* Dedicated to the memory of Louis Rapkine, a pioneer in these studies.
`201
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
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`

`202
`
`E. S. GIJZMAN B.4HKON
`
`D. Glutathione and Enzymic Reactions. . . . . . . . . . . . . . . . . . . . . .
`250
`1. Protection.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`250
`2. Regulatory Function, . . . . . . . . . . . . . . . . . . . . . . . .
`252
`E. Thiols and Oxygen Tension., . . . . . . . . . . . . . . . . . . . . . . .
`252
`F. Thiols and Cell Division and Growth. . . . . . . . . . . . . . . . . . . 253
`G. Thiols and Ionizing Radiations.. . . . . . . . . . . . . . . . . . . . . . . . . . .
`255
`VIII. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`257
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`258
`I. Introduction
`Thirty years have elapsed since Hopkins’ rediscovery of glutathione
`(152), and 20 years since Rapkine’s formulation of the role of thiols
`in cell division and cell growth. Although some progress has been
`made in these 30 years, we are still far from thoroughly understanding
`the functions of these thiol-containing substances. The universal
`distribution of glutathione in living cells and the reversibility of this
`oxidation-reduction system brought forth a large number of investi-
`gations on the determination of its oxidation-reduction potential, the
`mechanism of oxidation and reduction, and its possible’ function in
`cellular respiration. Yet all these investigations fell short of reaching
`their goal mainly because at the time they were made the peculiar
`sluggish nature of this oxidation-reduction system was not taken into
`consideration. It is certain that soluble thiols are one of the regula-
`tory mechanisms of cellular respiration. More progress has been made
`regarding the function of the -SH groups of the proteins. They have
`been found t o be essential for the activity of a large number of en-
`zymes; they have been found essential as binding posts between the
`protein and some prosthetic groups. A more detailed study of the
`reactivity of thiols has aided in the interpretation of the function of
`these two types of thiol compounds: the soluble thiols, of which gluta-
`thione is the most representative example, and the $xed thiol groups
`of the proteins. Far as we are from presenting a clear picture of this
`problem, the time is propitious for a discussion of the chemical reac-
`ttions of biological importance in which the thiol groups take part, of
`t.he functions of the soluble thiol compounds, the possible nature of
`the participation of the thiol groups in enzyme reactions and other
`physiological processes, and the participation of both soluble and
`fixed thiols in cell division, growth, and production of mutations.
`11. Some Properties of Thiols
`Thiols are extremely reactive substances. They oxidize and reduce
`rather easily, combine with a large number of halogen-containing
`
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`

`THIOL GROUPS OF BIOLOGICAL IMPORTANCE
`
`203
`
`substances in which the H of the -SH group is replaced by the halo-
`gen residue, and combine with a large number of heavy metals form-
`ing reversible mercaptides. They also combine easily with alde-
`hydes, quinones, etc. Some of these properties-those of physiological
`importance-will
`be discussed here, first in simple water-soluble
`thiols, and then in thiol groups fixed to the protein molecule.
`
`A. OXIDATION BY OXYGEN
`With the discovery of glutathione by Hopkins and the possibility
`that this thiol could play the role of electron transfer system from
`foodstuff t o molecular oxygen, the oxidation of thiols by atmospheric
`oxygen acquired importance. Mathews and Walker (204), who
`found that the oxidation of cysteine by atmospheric oxygen was in-
`creased in the presence of iron, believed that autoxidation occurred.
`The necessity of a metal catalyst for this oxidation was shown for
`the first time by Warburg and Sakuma (308) working with cysteine,
`and by Harrison (138) with glutathione. Thiols belong to the slug-
`gish type of oxidation-reduction systems, i.e., to those systems which
`are not easily oxidized by atmospheric oxygen, and the free energies of
`which cannot be determined by direct potentiometric methods.
`Oxidation of thiols by oxygen requires the presence of an electro-
`active oxidation-reduction mediator. The catalytic power of these
`mediators seems to depend on the oxidation-reduction potential of
`the catalyst, the position of the -SH groups in the thiol molecule,
`and the presence in the molecule of groups which may facilitate or
`hinder formation of a complex compound-the
`activated complex-
`between thiol and the catalyst. The influence of the ionic environ-
`ment has also to be considered (effect of cations, anions, pH). Further-
`more, the same metal may act as a catalyst with certain thiols and
`have no effect a t all with others. Thus, nickel is a powerful catalyst
`for tKe oxidation of HzS (Krebs, 177) and manganese for cysteine
`(Rosenthal and Voegtlin, 257), whereas nickel has no effect a t all
`on the oxidation of cysteine (Michaelis and Barron, 217), nor manga-
`nese on glutathione (Voegtlin et al., 304). Mercury oxidizes cysteine
`(Michaelis and Barron, 216), but has no effect a t all on the oxidation
`of glutathione (304). Furthermore, these catalytic effects may be
`depressed or enhanced by addition of other metals. The copper
`catalysis of thioglycolic acid oxidation in pyrophosphate buffer is
`depressed in the presence of manganese, whereas it is increased in the
`
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`

`204
`
`E. S. GUZMAN BARRON
`
`presence of iron; in carbonate buffer, on the other hand, copper and
`manganese are tremendously more active than apart (Kharasch
`et al., 174). Copper catalysis of cysteine oxidation is depressed by
`small concentrations of ferric salts, whereas larger concentrations in-
`crease the catalytic power (Baur and Preis, 44). Similar differences
`are obtained on measuring the effect of pH changes on the rate of
`oxidation. Copper catalysis of thioglycolic acid oxidation has an
`optimum a t pH 6 (175), but with glutathione oxidation the rate in-
`creased in direct proportion to the pH value, i.e., the rate of oxidation
`depended on the degree of dissociation of the -SH group of the glu-
`tathione (Lyman and Barron, 198). The catalytic action of these
`metals can also be greatly modified by the formation of metal com-
`plexes, as the Bernheims (47) have shown with the S-hydroxyquino-
`line complexes of copper, manganese, and vanadium in their effect on
`the oxidation of cysteine.
`The mechanism of this oxidation is not yet clearly understood,
`although Michaelis (211,213) greatly clarified the field, and proposed
`a theory which seems plausible. The postulated mechanism is based
`on the following facts: when a cobaltous salt is added to cysteine in
`the absence of oxygen there is formation of a cobaltous bis-cysteinate
`(I) of an intense blue-green color. On exposure to air it changes into
`
`S 1 \,p
`KR-*- / \,,-- - RH
`
`*-'Me--
`
`OH
`
`s
`
`s
`
`
`
`KR RK
`(1)
`(11)
`an olive-brown cobalti complex (11) which is stable (Schubert, 264).
`Similarly, in the presence of iron and the absence of oxygen ferrous
`co
`s ' s
`\!/
`l,?l
`
`R i R
`!
`
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`

`THIOL GROUPS OF BIOLOGICAL IMPORTANCE
`
`205
`
`ions combine with two molecules of cysteine, as in (I). The two
`hydrogen atoms of two RSH are replaced by one bivalent Fe atom
`and the two NH2 groups are attached to two more coordination
`places (111). There are two more coordination places available for
`the attachment of small molecules. In fact (111) combines with two
`molecules of CO to form the stable, crystalline compound (IV)
`(Cremer, 76). When oxygen is admitted to the solution of ferrous
`bis-cysteine there is immediate appearance of a deep indigo blue
`color, which soon disappears with the formation of cystine. This
`blue complex is probably due to the entrance of oxygen into the two
`coordination places left vacant in (111) to form a molecule similar to
`(IV). The complex, however, is unstable and immediately undergoes
`an intramolecular shifting of electrons. Since it is impossible to pre-
`pare this unstable complex, it is not known whether one or possibly
`two oxygen molecules are attached. The complex may be written:
`(V)
`Oxygen will withdraw one electron from Fe++ (according to the com-
`pulsory univalent oxidation theory of Michaelis (212)) and thus start
`an intramolecular chain of reactions, each step consisting in the trans-
`fer of one electron:
`Fe + +( RS); O2 ---+ Fe + + +( RS); 0,
`
`Fe + +( RS)*O2
`
`Fe+++(RS);O; - - -02Fe + + +(Rs
`--02Fe++(RS - R-S--H-R
`
`RS
`--02Fe+++<Rs- ---+
`
`RS
`
`Rs-
`RS
`--OzFe + +<Rs
`
`+ Fe++ + 0-;
`
`(VI)
`
`(VIII)
`
`( I X )
`
`In (VI) there is electron transfer from Fe++ to 02, in (VII) from RS-
`to 01, in (VIII) from RS- to Fe+++; in (IX) we have the formation
`of the stable oxidation compound R-S-S-R
`and reformation of
`the Fe++ catalyst. The HzOz formed in this process will oxidize
`another thiol, either alone or through iron catalysis.
`Aside from heavy metal catalysis, the oxidation of thiols by oxygen
`
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`

`206
`
`E. S. GUZMAN HARRON
`
`can take place with iron porphyrins as the catalysts, as shown by
`Krebs (177) for cysteine and by Lyman and Barron (198) for gluta-
`thione. The rate of oxidation of glutathione with hemin as a catalyst
`had a definite pH optimum a t pH 8, decreasing asymptotically
`toward both the alkaline and the acid sides of this p H value. A com-
`parison of the catalytic power of hemin and hemochromogens (iron
`porphyrin-nitrogen base complex compounds) revealed that this
`power depends on their oxidation-reduction potentials (Table I).
`Whereas the oxidation of cysteine by hemin was inhibited by HCN
`(178), that of glutathione was only slightly affected (198).
`
`TABLE I
`OXIDATION O F GLUTATHIONE BY ATMOSPHERIC OXYGEN WITH BLOOII HEMIN AND
`HEMOCHROMOGENS AS CATALYSTS'
`
`Catalyst
`
`PH
`7.4. . . . . . . Pilocarpine hemochromogen
`Hemin
`Nicotine hemochromogen
`Pyridine hemochromogen
`9.1. . . . . . . Pilocarpine hemochromogen
`Hemin
`0 Amount of hemin, 0.0002 mM;
`ture 27" (198).
`
`Time
`required
`for half
`oxidation,
`min.
`
`E, of
`catalyst,
`V.
`302
`-0.140
`108
`-0.138
`+O. 162
`80
`65.6
`+O. 146
`320
`-0.163
`1157
`-0.270
`amount of glutathione, 0.02 mM; tempera-
`
`R. OTHER OXIDIZING AGENTS
`Thiol compounds are easily oxidized by a variety