The Toxic Effects of Oxygen on Brain Metabolism and on Tissue Enzymes 2

Home , Choline oxidase, Succinic acid

Vol. 40 OXYGEN POISONING OF BRAIN TISSUE 171 Meyer, A. L. (1927). Amer. J. Phy8iol. 82, 370. Reiman, C. K. & Minot, A. S. (1920). J. biol. Chem. 42, 329. Michaelis, M. & Quastel, J. H. (1941). Biochem. J. 85, 518. Schales, 0. (1938). Ber. dtsch. chem. Ge8. 71, 447. Ochoa, S. (1939). Nature, Lond., 144, 834. Sollmann, T. H. (1942). Manual of Pharmacology, 6th -ed. Ochoa, S. (1943). J. biol. Chem. 151, 493. New York: Saunders. Penfield, W. & Erickson, T. C. (1941). Epilep8y and Cerebral Soskin, S. & Taubenhaus, M. (1943). J. Pharmacol. 78, 49. Localization. London: Baillibre, Tindall and Cox. Stadie, W. C. (1943). Personal communication. Peters, R. A. (1940). Personal communication. Stadie, W. C. & Riggs, B. C. (1944). J. biol. Chem. 154, 687. Potter, V. R. & Elvehjem, C. A. (1936). J. biol. Chem. 114, Stadie, W. C., Riggs, B. C. & Haugaard, N. (1944). Amer. J. 495. med. Sci. 207, 84. Quastel, J. H. (1939). Phy8iol. Rev. 19, 135. Still, J. L. (1941). Biochem. J. 35, 380. Quastel, J. H. (1943). Personal communication. Warburg, 0. (1930). The Metabolism of Tumours (trans. Quastel, J. H. & Wheatley, A. H. M. (1932). Biochem. J. F. Dickens). London: Constable and Co. 26, 725. Weil-Malherbe, H. (1938). Biochem. J. 32, 2257.

The Toxic Effects of Oxygen on Brain Metabolism and on Tissue Enzymes 2. TISSUE ENZYMES

BY F. DICKENS, National In8titute for Medical Research, London, N. W. 3, and Cancer Research Laboratory, Royal Victoria Infirmary, Newcastle-upon-Tyne (Received 23 October 1945) The evidence in Part 1 (Dickens, 1946) enables the present paper, some general evidence is collected following working hypothesis of the mode of action on the mode of inactivation by oxygen ofsome other of oxygen on brain metabolism to be advanced enzymes, particularly those which, like pyruvate with some confidence. oxidase, may play a part in brain metabolism. These (1) Exposure to unphysiologically high tensions ofoxygen experiments are of a preliminary and exploratory causes irreversible damage to brain respiration and gly- character, and serve mainly to define some con- colysis. ditions under which this type of inhibition may be (2) This damage is sustained by the enzymes themselves expected to occur, and to investigate some examples rather than by their coenzymes, as shown by the lack of of protection against oxygen poisoning in enzyme general protection in presence of excess of the more im- systems. portant coenzymes. (3) The following conditions tend to protect against The general nature of the inactivation oxygen poisoning: Presence of (a) glucose or (as shown in of enzymes by oxygen this paper) manganese ions, which protect succinoxidase in finely divided brain tissue; (b) ions of manganese, cobalt, A list has been compiled of enzymes known to be or magnesium (and probably calcium), which protect the sensitive to oxygen, or capable of inactivation by whole oxidative system in brain slices, with decreasing comparatively mild oxidants, and therefore open to activity in the order of the above series. the suspicion of being oxygen-sensitive under the (4) The brain enzymes poisoned by oxygen are mainly right conditions (Table 1). These observations were those concerned in carbohydrate metabolism. Oxidation widely scattered, and probably others could be ofglucose, fructose, lactate and pyruvate is inhibited, while the succinoxidase system, at least in intact tissue (slices) added: the only review found to bear on this subject, is much more resistant. There is support for the view that apart from the excellent one by Stadie, Riggs & the inactivation of an -SH component of the pyruvate Haugaard (1944) which appeared after this work oxidase system may be a primary stage in oxygen poisoning, was completed, is that of Hellerman (1937), which the resulting interference with resynthesis of adenosine is limited to hydrolytic enzymes. As this author triphosphate could then lead to general breakdown of rightly remarks: 'In most enzyme studies, the effect aerobic and anaerobic carbohydrate metabolism. of an ever present reagent, the oxygen of the air, (5) It is, however, not excluded that, in addition to the has been generally neglected.' pyruvate oxidase, another and earlier stage of carbohydrate There is another reason why such a list is in- metabolism might be inhibited, thus poisoning directly the oxidation of and fructose. evitably incomplete, and that is the enormous effect glucose on oxygen susceptibility of traces of contaminants, It is hoped later to study the effect of oxygen on especially -SH compounds and metallic ions, but components of the pyruvic oxidase system. In the also other substances less well defined. The following 172 F. DICKENS I946 typical experiences are quoted as excellent examples Table 1 (cont.) of the extraordinary complexity of the oxygen Enzyme poisoning of enzyme systems: nature* Enzyme class Authority B. Inactivation by exposure to oxygen under special cir- (a) Urea8e. Perlzweig (1932) showed that aeration of cumstances, or by mild oxidants: preservation by -SR crystalline urease weakened its action, and believed this compounds. (Less satisfactory evidence than the preceding.) Table 1. Enzyme8 8en8&tive to oxygen or to mild oxidants -SH (?) Myokinase Kalckar (1943) Enzyme -SH Transaminase Cohen, Hekhius & nature* Enzyme class Authority Sober (1942) A. Inactivation by exposure to gaseous oxygen (or air) -SH Triosephosphate Rapkine (1938), Rap- dehydrogenase kine & Trpinac (1939) (a) Proteolytic M Zymohexase (of Warburg & Christian -SH Cathepsin: tissue Bailey, Belfer, Eder & autolysis Bradley (1942) yeast)t (1942) -SH (?) Cerebrosidase Hellerman (1937, -SH Papain Hellerman (1937) p. 466) -SH Proteases, Peptidase Irving, Fruton & Berg- * mann Enzyme nature. -SH indicates evidence that an (1942) intact sulphydryl group is considered necessary for activity. (b) Hydrolytic ('deamidizing') X indicates that the enzyme either contains a metal as M Arginase Edlbacher, Kraus & part of its prosthetic group, or requires metallic ions for Leuthardt (1933), its activity. F indicates that the enzyme contains a flavo- Hellerman (1937) protein. (The first and second of these classes are often combined; both an -SH group and an activating metal -SH Urease Perlzweig (1932), Sum- being necessary. The classification of some of these enzymes ner & Poland (1933) cannot be considered as final.) (c) Fe-porphyrins t Zymohexase of animal tissues shows only inhibition by metallic ions, and is stable to oxidants (Herbert, Gordon, M Hydrogenase Steephenson & Stickland Subrahmanyan & Green, 1940). (1 1931), Hobermann & Rtittenberg (1943) to be due to removal of a sulphydryl component by oxida- M 'Pasteur enzyme' WEarburg (1926), Stern tion. The still more highly purified enzyme is, however, Melnick (1941) practically insensitive to the action of air, but is rendered M Catalase Mairks, G. W. (1936) sensitive again on the addition of minute amounts of copper (Eprolonged experi- salts (Hellerman, Perkins & Clark, 1933). maents) (b) Arginase behaves very similarly: cruder preparations M Cytochrome oxidase Diickens (this paper) are readily inactivated by oxygen, while purified ones are (d) Oxidases, etc. much less sensitive, or completely insensitive (Edlbacher, Pyruvic oxidase Bairron (1936), Mann Kraus & Leuthardt, 1933; Klein & Zeise, 1933, 1935). Quastel (1946), Arginase preparations are activated by metallic ions (Mn, D)ickens (1946) Fe, Co, Ni, Zn, Cd, and possibly others-cf. Richards -SH Succinoxidase Le,hmann, J. (1935), & Hellerman, 1940). Hellerman (1937) believes that the LiEebricht & Massart action of these metals is to form co-ordination compounds (19937), Bohr & Bean linking the enzyme molecule with that of the substrate. (19p40-1), Stadie (1943), (e) Pho8phoglucomutawe. This enzyme, which is necessary Diickens (this paper) for breakdown and synthesis of glycogen, is of peculiar -SH Choline oxidase Diickens (this paper) interest, since the present work has shown that at different of it can exhibit a wide variation of F Xanthine oxidase Di zxon (1925), Dixon & stages purification KCodama (1926) sensitivity to air. F d-Amino-acid oxidase StEladie (1943) Gill & Lehmann (1939) showed that there was a very slight increase in the amount of Robison-ester formed from F Cytochrome-c reduc- Haaas, Horecker & Hog- glycogen by muscle extract when the experiment was made tase ness (1940): (possibly) in absence of air. ? Fatty acid dehydro- Shapiro & Wertheimer Like many other oxygen-sensitive enzymes, phospho- genase (]1943) glucomutase is highly susceptible to the influence of traces -SH Triosephosphate de- Dickens (this paper) of metals; cobalt, magnesium and manganese strongly hydrogenase accelerate, nickel feebly so, and zinc retards the activity ? Formic acid dehydro- Gale (1939) of the enzyme. Concentrated solutions of reduced gluta- genase thione and oxidized glutathione respectively, accelerate, (e) Phosphorases and retard its activity (Cori, Colowick & Cori, 1938; Gill -SR Phosphoglucomutase Gill & Lehmann (1939), & Lehmann, 1939; Lehmann, 1939; and present work). Dickens (this paper) Although these properties suggest a direct connexion be- -SH (?) Neuberg ester phos- Engelhardt & Sakov tween oxido-reduction potential and activity, the relation- phorase (phospho- (1943) ship is, in fact, involved, e.g. substances such as insulin glucokinase) or caffeine are also inhibitory. The best way ofcircumventing V91. 40 OXYGEN POISONING OF ENZYMES 173 the experimental difficulties has in practice proved to be: of sensitivity of these enzymes on their physical (a) preparation where possible of the enzyme in a state state, as well as on the presence or absence of such that it is sensitive to oxygen; (b) study of conditions sensitizers or protective substances as already de- protecting enzymes of this kind against inhibition by scribed, it seems doubtful if this conclusion oxygen. Experiments devised with this end in view are is described below. justified on the available evidence. The data on oxygen inactivation of this enzyme are contained Table 1 shows that many different classes of in Table 2. enzyme are sensitive to oxygen. Although the Protection of sUccinoxidase against poisoning by presence of -SH groups, and/or metal activators oxygen is needed for the activity of many of these, some of the flavoprotein enzyme systems, which do not (a) By malonate. Malonic acid is a powerful require metals, are also sensitive. There is also competitive inhibitor for succinic dehydrogenase. evidence that oxidative inactivation of enzymes If, during exposure of the enzyme to oxygen, may sometimes be due to the sensitivity of quite malonate is present in concentration sufficient to different groups; for example, pepsin inactivation 'block' the active groups of the dehydrogenase by iodine may be due to iodination of tyrosine (C. 0-001M), and after exposure to oxygen the groups contained in the molecule of the enzyme, malonate is removed by washing, it is found that and oxidation of the phenolic group of tyrosine the enzymic activity has been completely protected may account for the inactivation of alkaline phos- (Table 3). The presence of malonate during the phatase by moderately strong oxidants (Sizer, 1942). incubation also protects the enzyme against auto- In general, however, the sensitivity ofthose enzymes lytic processes occurring in nitrogen, but these are which are weakened or inactivated by atmospheric less destructive of enzyme activity than the oxi- oxygen seems to be related to either: (a) destruction dative inactivation (Table 3); in some experiments of -SH groups by oxidation; (b) loss of catalytic there was little or no inactivation during incubation function of essential metals, presumably by their in nitrogen. Numerous other experiments, some conversion to higher valency forms; or (c) oxidative under H.O.P.,* also showed similar protection of destruction offlavoproteins. Examples of each type succinoxidase by malonate against oxygen poison- of inactivation are listed in Table 1. ing. This result is interpreted (see Discussion) to mean that the sulphydryl groups of the dehydrogenase are involved in inactivation by oxygen. EXPERIMENTAL The succinic dehydrogenase system may require (1) OXIDATIVE ENZYMES manganese for its activity (Massart, 1939), and if this is accepted as correct, the loss ofmanganese due A. Succinic oxidase system to oxygen might be pictured as occurring similarly This system has been more fully studied hitherto to that of manganese from arginase (Hellerman, in connexion with oxygen poisoning than any other 1937). The protection of succinoxidase activity (for references, see Table 1), and is known to be against H.O.P. by manganese, described below, seems oxygen-sensitive. This has been confirined, and the to be the first clear evidence of a possible function sensitivity of the components of the system oxi- of this metal in the succinic acid system. dizing succinate has been determined. These are (b) By manganese ions. In view of the effects of (a) cytochrome oxidase, (b) cytochrome c, (c) suc- metallic ions on the sensitivity of tissue respiration cinic dehydrogenase, and (d) a further component, to oxygen (see Part 1), similar experiments were which may be a flavoprotein (Axelrod, Potter & made with the succinoxidase system (Table 4). Elvehjem, 1942). The dehydrogenase itself is the Whereas manganese ions (0.00025M concentration) most sensitive of the components a, b and c (com- gave almost complete protection of succinoxidase ponent d cannot at present be studied separately); against oxygen, cobalt in the same concentration, with longer exposure to H.O.P. the cytochrome or magnesium even in sixteen times higher con- oxidase may also be slightly weakened (Table 2). centration, did not. In fact, even at this very low The sensitivity to oxygen of succinoxidase con- concentration, cobalt proved to be quite toxic, both tained in homogenized tissue has already been shown in oxygen and nitrogen. This illustrates further the (Dickens, 1946) to be greater than that of the great dependence of the effect of these metals on enzyme system when present in tissue slices. There the exact circumstances of the test, for cobalt ions are also wide variations in sensitivity from one protect the whole oxidation system in brain slices, enzyme extract to another (cf. Tables 2, 4). Bohr as well as the oxidation ofpyruvate (Dickens, 1946), & Bean (1940-1), who made a similar observation, but these ions are unable to protect the succinic consider that this may be related to the suscepti- system in homogenized brain tissue. On the other bility of the individual animals to oxygen, which hand, manganese ions protect both the total oxida- is known to vary widely. In view of the dependence * High oxygen pressure (see Dickens, 1946). 174 F. DICKENS I946

Table 2. Inactivation of succinic oxida8e 8ystem by oxygen (a) Preparation from pig-heart mu8cle by mechanical grinding of the washed tissue with sand, precipitation at pH 4-6 (cf. Straub, 1939), and resuspension in phosphate buffer at pH 7-4. 0-2 ml. enzyme/vessel; m/6 succinate; total vol. 3-0 ml. The enzyme was added from the side-bulb immediately after the first reading. Gas: air. Temp. 38°. Oxygen uptake (,ul.) in 10 min. after the following exposure of enzyme Nitrogen Oxygen Oxygen at 4-4 atm. h at 1 atm. , A Contents of vessel during test: None 24 hr. 3 hr. 3 hr. 2 hr. 3 hr. Enzyme alone (no succinate) 0-5 Enzyme+succinate (A) 102 100 78 51 41-5 30 Enzyme + succinate + meth. blue 106 93 85 66 49 Enzyme + succinate + meth. blue (0-03 %) 53 51-5 - 41-5 29 +HCN 0-002m (B) Enzyme + succinate + HCN (no meth. blue) 2 Enzyme +p-phenylene diamine (C) 63-5 63 - 57 - 42.5 As % of initial activities (mean results) after pre-treatment 1 atm. 4-4 atm. oxygen Nitrogen oxygen A' (3 hr.) (3 hr.) (2 hr.) (3 hr.) Total system (A, above) 90 50 41 30 Succinic dehydrogenase (B) 97 78 - 55 Cytochrome oxidase (C) 99 90 67 (b) From horse heart (a more resistant preparation than the above). 0-1 ml. enzyme/vessel. Activity with succinate measured in air. Temp. 38°. pH 7-4. Oxygen uptake (1u.) in 10 min. after following exposure of the enzyme

I~~ I Oxygen (1 atm.) Oxygen (4-4 atm.) A Nitrogen 1 , Control (2 hr.) (1 hr.) (2 hr.) (1 hr.) (2 hr.) 129 124 124 117 112 116 % of initial activity remaining: (100) 96 96 91 87 90

(e) Homogenized rat-brain tiS8ue (cortex). 40 mg. of moist tissue/vessel. Cytochrome c (0-1 ml.) added to give 2-2 x 10Mx solution. Oxygen uptake (pl.) in 10 min. after the following exposure of the homogenate Oxygen Nitrogen Air (1 atm.) (4-4 atm.) None (2 hr.) (2 hr.) (2 hr.) 56 46 35 30 Without added cytochrome: 21 - 8 Nitroprusside test (for -SH groups) in above after incubation: ++++ +++ i _ tion system of brain slices and also the oxidation of succinate by homogenized brain. Much further The role of oxaloacetic acid in oxygen poisoning of work would be needed to arrive at an explanation; succinoxidase at present one can only draw attention to the fact The succinoxidase preparations used in these that traces of metals have this profound influence experiments oxidized succinate only as far as on oxygen poisoning, and that under favourable fumarate and malate; the further stage of oxidation conditions they may give complete protection of of malate to oxaloacetate does not occur with them an otherwise susceptible enzyme. to any considerable extent, the accumulation of VoI. 40 OXYGEN POISONING OF ENZYMES 175 Table 3. Protection of succinic dehydrogenase against with acid. Swingle, Axelrod & Elvehjem (1942) the toxic effect of oxygen by malonic acid have shown that calcium ions greatly accelerate, the oxidation of succinate, their action being due Method. Rat-brain cortex, homogenized in M/30 phos- to the fact that these ions promote the breakdown phate, pH 7-4. of cozymase, thus preventing the formation of Centrifuged, supernatant fluid discarded, precipitate re-suspended in M/30 phosphate buffer. oxaloacetate. They found that the inhibition of 0-5 ml. of this suspension (equiv. to 100 mg. moist tissue) the enzyme by 0-2 ,uM-oxaloacetate was removed was incubated with the'addition ofeither 0-05 ml. phosphate to a large extent by the presence of glutamate. This buffer, or 0-05 ml. 0-1 M-sodium malonate, for 2 hr. at 380 was due to the removal of the oxaloacetate by its in either nitrogen or oxygen at 1 atm. interaction with glutamate, a reaction catalysed by After this incubation, the contents of the vessels were the enzyme transaminase. rinsed into centrifuge tubes with 4-5 ml. of M/30 phosphate, The possibility therefore arose that the oxygen centrifuged, supernatant discarded and the precipitate poisoning of succinic dehydrogenase might be rinsed into Warburg vessels with 2 ml. M/30 phosphate, caused by excessive formation or accumulation of 0-2 ml. cytochrome c, and (in the side-bulbs) 0-1 ml. oxaloacetic acid. M-sodium suceinate. Rates of succinate oxidation were Decisive evidence on this point measured at 1 atm. oxygen. has not been obtained; although some results may be considered to support it, there is also evidence Oxygen uptake of suceinic oxidase that other, irreversible, changes in the enzyme system (p1. 02/30 min.) occur during exposure to oxygen, especially when 2 hr. oxygen 2 hr. nitrogen the oxygen pressure is high or the exposure pro- longed. Period No With No With (min.) malonate malonate malonate malonate The influence of glutamate on the poisoning of sUC- 0-30 15-5 102 49 97 cinoxidase by oxygen 30-60 15 98-5 49 93 If oxaloacetate inhibition of succinoxidase pre- minute traces of oxaloacetate being strongly in- parations were responsible for the poisoning of the hibitory to succinic dehydrogenase. The accumula- dehydrogenase which occurs in oxygen, this poison- tion of oxaloacetate requires an active malic ing would be expected to be reversed by glutamate. dehydrogenase for which the presence of cozymase This, however, is only true as long as the trans- is necessary. Cozymase is rapidly broken down in aminase present is not also poisoned by oxygen, homogenized brain tissue, and, in the case of the for this enzyme is needed for the removal of muscle preparations used in the above experiments, oxaloacetate by glutamate. There is some reason most of the cozymase had been removed by washing to suspect that transaminase is, in fact, susceptible

Table 4. Action of metallic ions on the oxygen sensitivity of brain succinoxidase Method. Rat-brain cortex, homogenized in M/30 phosphate buffer, pH 7-4. Each Warburg vessel contained 40 mg. of moist tissue in a total volume of 2 ml. M/30 phosphate, together with 0-1 ml. cytochrome c and 0-05 ml. of the solution of metallic salt to be tested. The vessels were shaken at 380, while filled with either nitrogen or oxygen at 1 atm. After this incubation period, those vessels which had contained nitrogen were now filled with oxygen, and 0-1 ml. of m-sodium succinate was added from the side-bulbs. Readings of oxygen uptake were then taken as usual. Exp. i. Oxygen uptake (,ul/20 min.) after exposure to the following gases for 2 hr. at 380. Oxygen N[itrogen Metal present ... Na only Mn Mg Co Na only Mn Mg Co Concentration ofmetal 2-5 x 10-4 4 x 10-3 2-5 x 10-4 - 2-5 x 10-4 4 x 10-3 2-5 x 10-4 (molarity) 1. 02/20 min. 57 89 59 3-5 84-5 85 88 15-5 Activity after 2. 67 Activity after N2 % 105% 67 % (23%) Exp. ii. Protection by MnSO4 (2-5 x 10-4M) of a more resistant brain homogenate. Exposure times, 2 and 4 hr. at 1 atm. Gas Oxygen Nitrogen A Metal ...... No Mn +Mn No Mn ±Mn Exposure (hr.) 2 4 2 4 2 4 2 4 Activity (id. 02/30 min.) 52 39 72 68-5 65 55-5 78 73 As % of corres. control exp. 80 70 92 94 176 F. DICKENS I946 to poisoning by oxygen (see Table 1). This probably Table 6. Re8toration of succinoxidase activity after accounts, in part, for the fact that, in certain removal of added oxaloacetic acid, but not after experiments, some protective effect of glutamate poisoning by oxygen: failure to show OAA forma- was evident (Table 5, Exp. i), whereas in others it tion in oxygen was not. Rat-brain cortex homogenate. Centrifuge and re-suspend Table 5. Influence of glutamate on the poi8oning in M/30 phosphate, pH 7-4. Activity test: 0-1 ml. cyto- of succinoxidase by oxygen chrome c, 0-1 ml. M-succinate added. Exp. i. Protection by glutamate Exp. i. Lack of rever8al of oxygen poisoning on wa8hing Exposure of Oxygen Nitrogen Methods. Horse-heart succinoxidase preparation: 0-2 ml./ homogenate (1 atm., 2 hr.) (1 atm., 2 hr.) vessel (in side-bulb). Main part of vessels contained 0 3 ml. 0 2M-succinate, glutamate (01 ml. 0-2M) if so stated below, Un- Un- and phosphate buffer, pH 7 4, to total of 2 ml. Oxaloacetic After exposure Washed washed Washed washed acid (as sodium salt) was added in some vessels to illustrate Activity towards' the poisoning of this particular enzyme by this substance, succinate; pl. I 80 61 105 and the reversal by glutamate. oxygen uptake/ In some experiments the enzyme preparation, with or 30 min. wi'hout the addition of glutamate (M/30) was exposed to Ratio of activity: H.O.P. (4.4 atm. oxygen, 3 hr.), and the activity then tested Washed x 100 JAfte r nitrogen: 58 % as above. (OAA = Oxaloacetate.) Unwashed lAfte r oxygen: 50 % (ul./60 min.) Exp. ii. Reversal of oxaloacetate poisoning by washing of controls not exposed Homogenized brain system. Succinate as substrate. to H.O.P. Oxygen Oxygen With succinate only 237 uptake uptake Succinate + oxaloacetate, 8 (pI./ (P1./ 2 x 10-6M 10 min.) 10 min.) Succinate + glutamate (0.01 M) 412 (A) Without oxalo- 71 the same after 53-5 in addition to OAA acetic acid washing Succinate + oxaloacetate, 185 Oxaloacetic 13 ,,1 54 2 x 10-7M M/2500 (82 % in- Oxygen uptake hibition) (ALl./60 min.) Oxaloacetic 42-5 ,. .56 after 3 hr. M/10,000 (40 % in- exposure to Inactiva- hibition) Exposed to 2: 4-4 atm. 02 tion (B) Without oxalo- 35 ,, 21 Without glutamate 93 61% acetic acid With glutamate 382 7 % Incubated at 18 ,,~24 (during exposure and 37.50 with ox- during measurement) aloacetic acid M/2500 for Exp. ii. Lack ofprotection. The same enzyme preparation 1i hr. after long keeping in the refrigerator was no longer protected by glutamate against oxygen poisoning. Several Exp. fii. Lack of evidence of oxaloacetate accumulation other negative results were obtained with other preparations. in measurable amount Homogenized rat liver in M/30 phosphate buffer, pH 7-4 Reversibility of oxaloacetate poisoning, and non- centrifuged and resuspended, shaken in oxygen for 24 hr., reversibility of oxygen poisoning, of succinoxidase in Warburg vessel. Temp. 38°. After this period, the vessel contents were acidified by Whereas the poisoning of succinic dehydrogenase the addition of 0-5 ml. conc. citric acid solution, and 0 5 m'L. activity by oxaloacetate was readily reversed on of the aniline citrate reagent (Edson, 1935) was placed in removal of the oxaloacetate by washing (cf. the side-bulb. On tipping the reagent into the main part poisoning by malonate, Table 3), even after the of the vessel, no measurable decarboxylation occurred. enzyme had been exposed to oxaloacetate for more Hence no f-ketoacid had been accumulating under these than 1 hr., the poisoning of the same enzyme by conditions. This method would not detect with certainty oxygen was not so reversed (Table 6). Moreover, less than 4 x 10-5m-oxaloacetate, had this been present. no oxaloacetate was detectable by the sensitive The succinic oxidase activity of the same liver homogenate was inhibited by 51 % on the addition of 10-4m-oxaloacetic aniline-citrate test in a preparation of succinoxidase acid. which had been exposed to 1 atm. oxygen for 2-5 hr. Attempts made to reverse the oxygen poisoning Hence, if oxaloacetate formation is concerned in of succinoxidase preparations by incubation under the poisoning of succinic dehydrogenase by oxygen, anaerobic conditions, with or without the addition it can only partially explain the effect. of glutathione (cf. Hopkins & Morgan, 1938), were VoI. 40 OXYGEN POISONING OF ENZYMES 177 unsuccessful. Unlike malonate, pyrophosphate, Table 7. Reswitance of lactic dehydrogenase to H.O.P. which also is an inhibitor of succinic dehydrogenase, did not, in the one experiment made, protect the (1) In brain slices enzyme against the poisonous effect ofhigh-pressure Method. Slices of rat-brain cortex were incubated for oxygen. 2 hr. at 380: (a) in air at 1 atm., (b) in oxygen at 4-4 atm. B. Lactic Medium: phosphate-Ringer's solution containing glucose. dehydrogenase The slices were rinsed, and the oxygen-uptake rates The oxidation of lactate by tissue preparations measured in presence of lactate, cozymase, HCN, and is inhibited by oxygen (Dickens, 1946), but con- methylene blue (Method i). Alternatively, the lactate sideration of the mechanism of the oxidation dehydrogenase activity after exposure was measured anaerobically in presence of ferricyanide, HCN, and cozy- shows how complicated the interpretation of this mase by the method of Quastel & Wheatley (1938) fact may be. In fact, lactic dehydrogenase itself (Method ii). is highly resistant to oxygen, as the following experiments show. Method i. Air-incubated 4-4 atm. oxygen- slices incubated The activity of lactic dehydrogenase in slices of rat-brain -' cortex, after 2 hr. exposure to 4-4 atm. of oxygen, was With With compared with that of slices kept in air at 1 atm. There lactate lactate was no loss of activity of the dehydrogenase (Table 7). Blank added Blank added Other experiments confirmed this result (Table 7). Oxygen-up- 1-3 2-4 1i1 2-8 take rate There is, therefore, no evidence to suggest that (Qo2) Without With lactic dehydrogenase is at all sensitive to oxygen. Method ii. With cozymase cozymase This agrees with observations reported to us by 12ictate ,1 k--, Stadie (1943). The poisoning of lactate oxidation in ax,nd cozy- - Lac- + Lac- - Lac- +Lac- mase tate tate tate tate tissues (Dickens, 1946) is probably accounted for CO2 evolution 4-8 1-65 2-85 1-9 5.4 by injury to other parts of the complex system by rate (Qco2) which lactate and pyruvate are metabolized in intact tissues. (2) In purified lactic dehydrogenase solution Method. Dialyzed extract of acetone muscle powder C. Malic dehydrogenase (cf. Green, Needham & Dewan, 1937). Pig-heart flavo- This enzyme was tested with the same prepara- protein of Straub (1939), cozymase prepared according to Williamson & Green (1940). Care was taken to use minimal tion of lactic dehydrogenase from rabbit muscle, amounts of dehydrogenase to secure maximum oxygen and under the same conditions, as the lactic uptake; by avoiding any excess of this enzyme it becomes dehydrogenase described above. The results of the factor limiting the rate of the reaction, hence any exposure to 4-4 atm. oxygen for 3-25 hr. showed destruction of the enzyme would be clearly revealed. no destruction, as compared with a portion of the Each vessel contained: 0-5-0-05 ml. enzyme, 0-2 ml. enzyme incubated in nitrogen, and negligible loss M-lactate, 0-8 mg. cozymase, 0-4 ml. M-HCN, 0-3 ml. flavo- of activity in either nitrogen or oxygen as compared protein solution, and sufficient 0-05M-phosphate, pH 7.4, with a control portion kept at 00 (Table 8). This to make the total volume 3-0 ml. 0-2 ml. methylene blue dehydrogenase, like lactic dehydrogenase, is in our (0-5 %) was added from the side-bulb at the beginning of the readings of oxygen uptake. experience quite stable to oxygen, though according to Barron (1943) it is an -SH enzyme. Pre-treatment of enzyme On ice In nitrogen In oxygen 380, 3j hr. 4-4 atm. Table 8. Malic acid dehydrogenase 380, 3j hr. Volume of enzyme solution, 0-5 ml./vessel: Method: same as for lactic dehydrogenase (Table 7) but Rate of oxygen uptake 103 103 with sodium l-malate as substrate instead of lactate. Each (pl./30 min.) vessel contained 0-5 ml. enzyme, 0-8 mg. cozymase, 0-3 ml. Loss of activity in oxygen 3-5 % heart flavoprotein, 0-2 ml. 1-malate (M-solution), 0-4 ml. M-HCN, and M/20 phosphate buffer, pH 7-4. Total vol. 3 ml. Volume of enzyme solution, 0-05 ml./vessel: Oxygen uptakes were measured manometrically, im- Rate of oxygen uptake 214 mediately following the addition from the side-bulb of (,ul./70 min.) 0-2 ml. methylene blue (0-5 % solution). Loss of activity in oxygen 0 % Pre-treatment of enzyme Onice In nitrogen In oxygen (Volume of enzyme 0-01 ml.: oxygen uptake in control 380, 31 hr. 4-4 atm. experiment falls to 81 ,ul./70 min., showing that excess 380, 31 hr. enzyme was not used, particularly in latter of above Rate of oxygen uptake 54-5 53 53 experiments.) (,ul./30 min.) 178 F. DICKENS I946 D. Triosephosphate dehydrogenase Table 9. Triosephosphate dehydrogena8se The activity of this enzyme system was followed Method: see text. 1 ml. muscle extract, 02 ml. M/2 by the rate of the dismutation: sodium pyruvate, 0*3 ml. M/2 sodium bicarbonate, 0 7 ml. M/5 sodium fluoride, 1 mg. cozymase, and water to make Triosephosphate + pyruvate--phosphoglyceric acid +lactate the final volume 3 ml. The side-bulb contained 0 3 ml. c. (ef. Green, Needham & Dewan, 1937; Rapkine, 1938; M/10 hexose diphosphate solution, and the carbon dioxide Rapkine & Trpinac, 1939). evolution following the addition of this to the main part This reaction has the same rate in nitrogen as in oxygen of the vessel was followed manometrically. Temp. 380. (Green et al. 1937 and Corran, Green & Straub, 1939). After Gas-space of manometer vessels contained 5 % (by vol.) removal of cozymase by adsorption on charcoal from the of carbon dioxide. enzyme, the latter is more sensitive to oxidants (Rapkine, 1938) andcozymase canprobably protect this enzymeagainst (a) Enzyme preparation not freed from cozymase oxidation. Rapkine & Trpinac (1939) showed that the in- Incu- activation of the enzyme by various oxidizing agents could Incu- bated be reversed by subsequent contact with reducing agents. bated 3 hr. Un- 3 hr. in 4-4 atm. In studying the effect of oxygen on this enzyme, Pre-treatment ofenzyme treated nitrogen oxygen advantage has been taken of the above interesting Activity of dehydro- properties. It was found that the triosephosphate genase (,Il. C02/15 min.) dehydrogenase is susceptible to oxygen poisoning, 1. (No NaF added) 445 586 536 2. (In presence of 597 572 but only becomes sensitive to this action after the NaF) cozymase has been removed. Table 9 shows that Percentage of control in N2, after H.O.P.: 91 %, 96 % before removal of cozymase, exposure to 4-4 atm. respectively. of oxygen for 3 hr. caused only 4-9 % poisoning. (In the second of these experiments, the enzyme solution After removal of cozymase by treatment with was neutralized to pH 7-4 but not in the first. This probably charcoal, a similar exposure to oxygen caused 24 % accounts for the apparently slightly higher activity in inactivation. presence of NaF.) It is to be noted that the sulphydryl groups of (b) Enzyme treated with charcoal to remoye this enzyme preparation are rather insensitive to cozymase almost completely oxygen; a weakly positive nitroprusside test, of 2-0 g. acetone powder from rabbit skeletal muscle, sus- about the same strength, being observed after pended in 24 ml. distilled water and dialyzed at 0° against exposure as above to either nitrogen or H.O.P. distilled water for 24 hr. Volume now 30 ml. The clear Pr'obably only a small part of this reaction was due centrifugate was shaken twice with 1 g. washed charcoal to the -SH groups of the enzyme itself, the re- (Merck's Medicinal Ultra-carbon) each time, centrifugation mainder being due to contaminants. Specially made being used to remove the charcoal. After neutralizing the control tests gave no support to the idea that traces solution to pH 7-4 with 0-16 ml. N-NaOH, and keeping of metals introduced by the treatment with charcoal at 00 for 2 days, a protein precipitate formed which carried (which always contains metals) could have brought down completely all residual traces of suspended carbon. about the sensitization of the enzyme to oxygen. The centrifuged solution (23.5 ml.) was then perfectly was clear. Other conditions as for (a) above: NaF was present All charcoal removed by centrifugation before in the activity tests. the test was made, so that its presence was not Activity of dehydrogenase (,ul. C02/15 min.) measured responsible. in presence or absence of added cozymase. Pre-treatment of enzyme: E. d-Amino-acid oxidase, flavin-adenine- and Incubated at Incubated at dinucleotide, diaphora8e 380 for 3 hr. in 380 for 3 hr. in Although d-amino-acid oxidase is not present in Kept on ice nitrogen 4-4 atm. oxygen 11 brain tissue, which oxidizes only glutamic acid Cozy- Cozy- Cozy- Cozy- Cozy- Cozy- among amino-acids, it is of interest (a) as a flavo- mase mase mase mase mase mase protein, a class which includes oxygen-sensitive absent present absent present absent present (or at least H202-sensitive) enzymes (Table 1), and 2-5 41 5 41 3 31 (b) because it was stated by Stadie (1943) to be highly Inactivation: 0% 24% susceptible to oxygen, when used in the form of homogenized tissue suspensions, but very resistant of these preparations was very high, even when when used as tissue slices. they were maintained in pure nitrogen. Hence Our own experiments with homogenized kidney comparison with the rate of destruction in oxygen tissue were unsatisfactory as evidence of the was unreliable. After even 30 min. incubation at stability or otherwise towards oxygen of this 38° in either nitrogen or oxygen the enzyme was enzyme. This was because the rate of loss of activity inactivated, the rate of oxygen uptakes being then Vol. 40 OXYGEN POISONING OF ENZYMES 179 almost equal whether alanine was added as sub- Although brain tissue does not contain this strate or not. In view of this result, the system enzyme, it does contain the same prosthetic group in homogenized tissue was not further studied. (flavin-adenine-dinucleotide; abbreviated F.A.D.). Instead, the behaviour ofpurified enzyme extracts This is the prosthetic group of other important was examined. The results (Table 10) showed that flavoproteins present in brain. Consequently, the the enzyme separated from autolytic kidney en- F.A.D. content of brain tissue has been examined zymes is stable in both nitrogen and oxygen. The after exposure to H.O.P. Table 10. d-Amino-acid oxidase Slices of fresh brain cortex were cut as usual. These were weighed into phosphate-Ringer's solution containing glucose Preparations: (2.5 ml. portions) so that each tube of the 'pressure-pot' (A) Crude extract. This was made from acetone-dried apparatus contained one good slice of about 70 mg. moist pig kidney by the method of Warburg & Christian (1938), weight. The slices were incubated for various periods in and consisted of the centrifuged aqueous extract. either air at 1 atm., or pure oxygen at 3-5 atm. (B) Purified extract. Method of Negelein & Bromel At the end of the incubation, the slices, which were quite (1939) carried as far as their 'Stage B'. The solution con- intact, were carefully removed to centrifuge tubes, 1 ml. tained approximately 2-5 mg. protein/ml., and 0-2 ml. of water was added to each, and the whole heated to 800 (c. 0-5 mg. protein) was used for each vessel. Both solutions for 15 min. to liberate the F.A.D. were water-clear. The F.A.D. content of the liquid after centrifugation was Method. The rates of oxygen uptake with dl-alanine as estimated by measurement of the oxygen uptake produced substrate were measured at 380 in air, using the Warburg by addition of 0 4 ml. of the solution to a preparation of apparatus. Each vessel contained the enzyme (0 5 ml. amino-acid oxidase which had been freed from F.A.D. by prep. 'A' or 0-2 ml. 'B'), 1-0 ml. m/10 pyrophosphate buffer acid precipitation (cf. Ochoa & Rossiter, 1939). Alanine of pH 8-3, water to make the total volume 2 ml. 0-2 ml. was added as substrate. M/10 or M/20 alanine solution was added at the beginning The results are expressed for convenience as ,ug. of of the readings, from the side-bulb of the vessels. F.A.D./g. moist brain tissue. The relative values are correct, Results: but the absolute amounts are only approximate, no standard preparation of r.A.D. being available. Instead of this, we Preparation A. Without added alanine, oxygen uptake have assumed that, as found by Ochoa & Rossiter (1939), negligible (1 jul./10 min.). an oxygen uptake of 180 ,il./30 min. would correspond with Pre-treatment of enzyme solution: the presence of 1 ,Lg. F.A.D. This assumes that the After enzyme After 3 hr. at used was of similar activity to theirs; an assumption at Control 3 hr. at 380 in least approximately correct. kept on 380 in 4-4 atm. The results (Table 11) showed no destruction of ice nitrogen oxygen the prosthetic group under these conditions. Enzyme activity 26 23 23 (pi. 02/10 min.) Percentage of ice 89% 89% Table 11. Flavin-adenine-dinucleotide content control of brain tissue

Inactivation due - - 0% to H.O.P. Pre-treatment of brain slices: Air, 1 atm., Oxygen 3-5 atm., Preparation B. Without added alanine, oxygen uptake None 380 for 380 for nil. (fresh Enzyme activity after pre-treatment as above: tissue) 2-25 hr. 4 hr. 2-25 hr. 3 hr. 4 hr. After After Flavin-adenine- Control 3 hr. at 3 hr. at 380 dinucleotide kept on 380 in in 4-4 atm. content (,ug./g.) ice nitrogen oxygen 14-1 13-0 13-1 12-3 13-3 12-9 55-5 55 55 (0d. 02/10 min.) As a further example of a flavoprotein enzyme, (half-quan- that extracted from pig's heart (Straub, 1939) has tity of enzyme been studied coenzyme =27 Iul. 02) (diaphorase, factor). Inactivation Nil Nil Diaphorase was made from pig's heart (Straub, 1939). The purification was carried as far as the elution from Cy fairly crude extract was inactivated to the extent alumina. It was a clear yellow solution in phosphate of 1-1 % after 3 hr. at 380 either in nitrogen, or in buffer, pH 7-4, and was stored at 00. oxygen at 4-4 atm. Further purified, it was com- For testing its activity, a dialyzed aqueous extract of acetone made from rabbit skeletal muscle was pletely stable under the same conditions of exposure powder used (cf. Green et al. 1937; Corran et al. 1939). The rates of oxygen to E.O.P. or nitrogen. These experiments do not, uptake produced by the addition of various volumes of therefore, provide any evidence that this enzyme the diaphorase solution to this test system were found to is sensitive to oxygen. Barron (1943) states that be strictly proportional to the volumes added; hence. any this is an -SH enzyme. change of activity in the diaphorase would be clearly Biochem. 1946, 40 12 180 F. DICKENS I946 revealed by the activity tests. The activity ofthe diaphorase are either themselves inactivated by H202 which was such that addition of 0 3 ml. caused an oxygen uptake accumulates, or they may inactivate other enzymes of 59 pl./5 min. with the test system. present, through the same mechanism. If catalase The diaphorase was incubated in nitrogen at 1 atm. or were to be destroyed in brain tissue by exposure in oxygen at 4-4 atm. for 2-5 hr. at 380. Activity tests upon these samples and upon the unincubated diaphorase were to H.O.P., other brain enzymes might also succumb. then made. For these tests, each Warburg vessel contained: The estimation of catalase activity was as described by 0 3 ml. diaphorase (in the side-bulb), 0-2 ml. M-sodium Fujita & Kodama (1931). This method gives rather variable lactate, 0-8 mg. cozymase, 0 4 ml. m-HCN, 0-2 ml. methy- results. It was especialy noticed that after shaking brain lene blue (0 5 % solution), and water to a total volume of tissue slices in Ringer's solution, part of the activity passed 3 ml. The measurement of oxygen uptake was made in into the solution. As the activity of catalase in brain tissue air at 380. is very low, while that of blood is high, this was probably due to slow washing out of traces of blood remaining in This flavoprotein was not at all inactivated by the tissue during slicing. The order of accuracy of the 2-5 hr. exposure to 4-4 atm. of oxygen at 38° results is therefore only sufficient to detect a considerable (Table 12). inactivation of catalase. Table 12. Diaphorase (heartflavoprotein, Test of this point with brain cortex did not 'coenzyme factor') yield evidence of catalase inactivation. The results (Table 13) make it unlikely that destruction of Pre-treatment of enzyme solution: catalase, if it occurs, plays any important part Nitrogen, Oxygen, None 2-5 hr. at 380 2-5 hr. at 38° in the toxic effect of H.O.P. on brain metabolism. (kept on ice) 1 atm. 4-4 atm. Exp. iii of Table 13 shows the catalase content Oxygen uptake rates of test system (4il. 02/5 min.): of the tissue after it has been shaken for 3*5 hr. in 61 61 68 Ringer's solution, removed from the solution, and the activity of the tissue alone (i.e. without the On the whole, therefore, the evidence presented catalase washed into the solution) determined: here is against any great oxygen-susceptibility of these experiments are marked 'tissue only'. Com- these flavoprotein systems. It is, of course, probable parison of the activity shown in these experiments, that some flavoproteins are oxygen-sensitive (see in air or oxygen at 1 atm., with those of Exp. ii Table 1), and it certainly cannot be maintained under H.O.P. (where the tissue was similarly trans- that a negative result with one particular prepara- ferred to fresh Ringer's solution for the measurement tion excludes the possibility that other preparations of catalase activity) show that there is no greater of the same enzyme may be inactivated by oxygen. fall with H.O.P. than in air or oxygen at 1 atm. More accurate results are unobtainable with brain F. Catalase tissue, for the reasons mentioned above. (The relevant figures are italicized in Table 13.) In the absence of catalase, enzymes such as Stadie (1943) also reports that purified prepara- xanthine oxidase which form hydrogen peroxide tions of catalase were highly stable to H.O.P. Table 13. Catalase content of brain tissue During the exposure to oxygen, the slices of rat-brain cortex were suspended in phosphate-Ringer's solution containing glucose. Brain no. Pre-treatment of brain slices (Qcatalase) None (fresh tissue). Unwashed tissue 73 ii 3-5 atm. 02: 75 min. 62 Ditto Tissue onlv (removed 33 from suspension fluid) 3-5 atm. 02: 150 min. dupl. 39 3-5 atm. 02: 210 min.. dupl. 36 iii None. Unwashed dupl. 56 The same. Shaken 210 min. in air (1 atm.) JSoln. + tissue 51 iTissue only 32 The same. Shaken 210 min. in 02 (1 atm.) {Soln. + tissue 61 lTissue only 26 VOI. 40 OXYGEN POISONING OF ENZYMES 181 contained 0-85 mg. of pyrophosphate-P. The gas space of G. Choline oxidase the vessels contained nitrogen with 5 % (by vol.) carbon This is an enzyme which is stated to depend for dioxide, and the temperature was 380. The activity curve groups, and was of the hexokinase preparation tested under the same con- its activity on intact sulphydryl ditions, showed that 0.5 mg. hexokinase gave maximum tested because so many other -SH enzymes are activity (140 ,ul. C02 liberated in 20 min.). The basis of sensitive to oxygen. the manometric method of following the reaction rate is The preparation of the enzyme was based on the work of the liberation of one extra phosphoric acid group, which Mann & Quastel (1937) and Mann, Woodward & Quastel occurs in the reaction: (1938). The liver of a rat was homogenized in 01IM-phos- Glucose + adenosine triphosphate = phate, pH 7*4, and the suspension strained through muslin. glucose-monophosphate + adenosine diphosphate. 1 ml. of extract was equivalent to 0 5 g. of fresh liver. This acidic group liberates 1 mol. C02 from the NaHCO3. Portions of the extract were shaken in air, or in 4-4 atm. The aqueous hexokinase solution, plus various amounts pure oxygen, at 38° for 180 min. The activity of the choline of glucose, was incubated at 380 in nitrogen or oxygen, as oxidase was then estimated by the rate of oxygen uptake stated, and afterwards the activity of the enzyme was which occurred on addition of choline. determined as above. The activity remaining after exposure For the estimation each vessel contained: enzyme 0 5 ml., is expressed in terms of the weight of original enzyme which water to make the total volume 2 ml., and in the side-bulb would be required to show the same activity as that 0-2 ml. of a solution containing 4 mg. choline chloride in observed; the latter being read from the weight-activity 0-1M-phosphate buffer, pH 7-4. Temperature 380. Gas curve. space, air. No evidence was obtained that it was inactivated This proved to be another example of an oxygen- by oxygen (Table 15). In the absence of glucose sensitive -SH enzyme, and was inactivated to the the enzyme is inactivated rapidly (Dixon, 1940), extent of 60 % by 3 hr. exposure to 4*4 atm. of hence glucose had to be present as stabilizer. It is oxygen (Table 14). therefore not possible to say what its sensitivity to oxygen in absence of glucose might be. Table 14. Choline oxidase Pre-treatment of Table 15. Hexokina8e enzyme 0 3 mg. hexokinase originally taken. Wt. Air, Oxygen, of hexokinase (mg.) 1 atm., 4-4 atm., after 3 hr. 3 hr. remaining Oxygen uptake With choline 298 152 Exp. Glucose Period of N2 02 02 (,ul./150 min.) iNo choline 54 56 no. conc. exposure (1 atm.) (1 atm.) (4 4atm.) Increase due to choline 244 96 I M/5 0-5 hr. 0-26 0-28 -

M/100 l,, 0-22 0-25 - Inhibition due to H.O.P. 61 % M/500 0-17 0X19 1,, NoN-OXIDATIVE ENZYMES II M/500 0-17 0-13 (2) M/1000 015 0-12 The above enzymes (A-G) are all oxidative in M/5000 006 003 - character. Two examples only have been studied III M/1000 0-11 0-05 - M/2000 002 005 of non-oxidative enzymes-hexokinase and phos- 1,, phoglucomutase-both of which are believed to be M/4000 0-00 0.00 of metabolism. IV m/250 0-18 0-16 - primary importance in carbohydrate M/500 0-14 0-16 The former, although an -SH enzyme (Dixon, M/1000 0-10 0-11 - 1940), was not inhibited by exposure to oxygen; V m/5 3 hr. 0-25 0-24 but ill-defined the latter was, only under certain m/25 ,,~ 0-19 0-20 conditions of purity. M/100 0 14 - 0-14 No glucose added: H. Hexokinase VI 0 mmin. 0-04 0-00 10 min. 0-00 0-02 This enzyme, prepared according to Meyerhof (1927) and Colowick. & Kalckar (1941), was obtained from baker's yeast as a white powder, completely soluble in water, of I. Phosphoglucomutase which 0 3 mg. was sufficiently active to give vigorous Evidence suggesting that this enzyme might be phosphorylation of glucose. to oxygen, even to atmospheric air, was For tests of activity each vessel contained 0-3 mg. of sensitive hexokinase, 0.5 ml. of M-glucose, 0-1 ml. 1 % MgCl2, 0-8 ml. presented by Gill & Lehmann (1939) and Lehman 1-3 % NaHCO3, water to make the total volume 3-0 ml. (1939). The former authors report one experiment The solution of adenosine triphosphate (0-2 ml.) was added in which the Robison ester formation from glycogen from the side-bulb at the beginning of the readings; it was 8 % less in air than in vacuo after 1 hr., and 12-2 182 F. DICKENS 1946

16 % less after 2 hr. of enzyme action. This enzyme pH 5-2, and heating to 520 for 30 min. The clear colourless system has since been separated from the crude filtrate was adjusted to pH 7.5, again filtered, and dialyzed muscle extract, as used by Gill & Lehmann, and at about O° against distilled water for 5 days. The solution was concentrated by freezing, to 19 ml. (Extract C). its action has been cleared up by the work of Cori In addition, a further precipitate formed only slowly and co-workers. (overnight) on the addition of the ammonium sulphate in It was shown by Cori et al. (1938) and Gill & the last stage, just described. This was worked up separately Lehmann (1939) that phosphoglucomutase is prob- as just described (Extract D), but was of lower activity ably an -SH enzyme and is highly susceptible than the main extract (Extract C). to the influence of metals. Magnesium, manganese, Cori ester. The potassium salt of this ester (glucose-l- cobalt and nickel greatly accelerate the action, phosphoric acid) was made from starch with the aid of while zinc (except in extremely low concentration, potato juice as described by Hanes (1940). Found: 5 min. hydrolyzable e.g. 10-7M, when it may be accelerative), copper 'Apparent inorganic P'=0-16 %. P= 7-96 Calc. 0 % and 8-33 % respectively. and iron salts are inhibitory. The electrodialyzed %. Method of experiment. 5 ml. portions of the aqueous extract is practically inactive without added metals solution of the potassium salt of glucose-l-phosphate were (Cori et al. 1938). Gill & Lehmann (1939) and measured into the vessels, and warmed to 380. The solution Lehmann (1939) showed that caffeine, oxidized contained approx. 1 mg./ml., the exact content being deter- glutathione, various oxidizing agents or insulin mined by phosphorus analyses. inhibited the action of the enzyme, while reduced The enzyme solution was added at the beginning of the glutathione, and other reducing agents accelerated experiment (0 min.). The experiments at 1 atm. were done it. The action of insulin was ascribed by Cori et al. in Warburg vessels; those at H.O.P. in 'pressure pots' (1938) to its zinc content, but this is denied by (see Dickens, 1946). After the desired incubation times, the reaction was stopped by the addition of 2 ml. 10 % who observed a similar Gill & Lehmann (1939), trichloroacetic acid. Blanks were run on the enzyme and inhibition with insulin hydrochloride of very low Cori ester alone. Analyses were made of: (a) 'apparent zinc content. inorganic P'; (b) easily hydrolyzable P (5 min. at 1000 in These properties appear to have some relation- N-sulphuric acid). The latter represents the unchanged Cori ship to the effect of some of the substances named ester. above on oxygen convulsions, as observed by H. P. A control experiment showed that, without the addition Marks (1944); hence they have been cited in some of enzyme, the Cori ester was perfectly stable at 38° under detail. For our experiments, the purification of the the conditions of incubation adopted. enzyme was by the method of Cori et al. (1938); followed by removal of other associated enzymes At first, when the crude undialyzed muscle according to Colowick & Sutherland (1942). As extract made according to Cori et al. (1938) was already mentioned the oxygen sensitivity of this used, oxygen had no effect on the activity (Table 16). enzyme is greatly dependent on the method and After dialysis, the extract became oxygen sensitive. stage of purification. After further purification by the method of Colowick & Sutherland (1942), at various stages up to full Undialyzed extract. One large rabbit was killed, the purification by this method, the enzyme was still abdominal aorta exposed, and about 600 ml. warm 0 9 %/ to NaCl was forced through to remove the blood, via the clearly sensitive to oxygen. The sensitivity abdominal vein, from the hind limbs. The muscle of both oxygen reached a maximum in the partially purified, hind limbs was excised, placed in ice, and passed twice freshly prepared enzyme. This was especially true through the ice-cold mincing machine: wt. 370 g. This of the immediate precipitate which occurred on mince was ground well with 370 ml. ice-cold distilled water, adding ammonium sulphate to the solution freed kept at 0° for 2 hr. and the extract expressed through from phosphorylase (Colowick & Sutherland, 1942). cheese-cloth. A second similar extract was combined with A portion of this precipitate which came down only the first; total volume, Extract A, 805 ml. after standing for some hours, was much less sensi- Dialyzed extract. 630 ml. of the above extract were lower dialyzed in cellophane sausage-skins at 12° for 5 hr., then tive to oxygen, and was also of much activity. for 16 hr. at 30 against 5 1. distilled water. After filtering On keeping the highly oxygen-sensitive preparation from the precipitated globulins, the filtrate was used in the cold room, it gradually deposited a white (Extract B). Inorganic P =413 mg./100 ml.; easily hydro- precipitate containing protein (cf. Cori et al. 1938). lyzable P (5 min. at 1000 in N-sulphuric acid) =0-73 mg./ This was removed by centrifuging, when the super- 100 ml. (cf. Cori et al. 1938). natant clear solution, although still highly active Purification ofdialyzed extract (cf. Colowick & Sutherland, as phosphoglucomutase, was no longer sensitive to 1942). 530 ml. above extract were made 0 4 saturated with oxygen. These results are collected in Table 16. ammonium sulphate and warmed to 300. The precipitate Their general significance has been discussed in the (phosphorylase) was removed; the clear solution contained the phosphoglucomutase together with isomerase. The Introduction. latter was removed by precipitating the mixture with an In an attempt to re-sensitize the highly purified addition of half the previous amount of ammonium sul- enzyme to oxygen, many experiments were made phate, resuspending the precipitate in veronal buffer at with metal-activated enzyme, comparing the ac- VoI. 40 OXYGEN POISONING OF ENZYMES 183

Table 16. Pho8phoglucomutase Undialyzed enzyme (Extract A) Amount of enzyme used: 0.1 ml./5 ml. Cori ester. P analyses: mg. P/vessel A - r 'Apparent Easily % conversion inorg. P' hydrol. P of Cori ester Initial. Enzyme only (0.1 ml.) 0-056 0 Cori ester (5 ml.) 0*026 0 359 Total 0-082 0359 Incubated approx. 2 hr. at 380: Exp. I Oxygen 4-4 atm. 0-096 0-121 66 Air 1 atm. 0-094 0-123 66 Exp. II Oxygen 1 atm. 0 095 0-083 77 Nitrogen 1 atm.* 0 095 0-082 77 Incubated 1 hr. at 38°: Exp. III Oxygen 1 atm. 0-069 0-163 55 Nitrogen 1 atm.* 0-066 0-151 58 Undialyzed enzyme is practically insensitive to oxygen. Dialyzed enzyme (Extract B) Initial. Enzyme only (0-1 ml.) 0-005 0 Cori ester (5 ml.) 0-026 0-359 Total 0-031 0.359 Incubated approx. 2 hr. at 380: Exp. IV Oxygen 4-4 atm. (Freshly made enzyme) 0-025 0-136 62 Air 1 atm. ,, 0-026 0-140 61

Exp. V Oxygen 1 atm. VP 0-025 0-102 71-5 Nitrogen 1 atm.* ,, 0-025 0-021 94 Exp. VI Oxygen 1 atm. (Kept 2 days) 003 0-114 68-5 Nitrogen 1 atm.* ,, 0-03 0-074 80 Exp. VII Oxygen 1 atm. (Kept 5 days) 0-02 0-169 53 Nitrogen 1 atm.* ,, 0-02 0-134 63 The freshly prepared dialyzed enzyme is highly susceptible to oxygen; though H.O.P. is no more inhibitory than is air at 1 atm, In nitrogen, however, the activity is much higher. The enzyme on keeping loses some of this susceptibility to oxygen, although the effect is still quite evident after 5 days. Enzyme purified by ammonium 8ulphate, etc. (Extract C) Amount of enzyme used: 0-1 ml. of 1 in 10 dilution (=0-01 ml. Extract C). This amount of enzyme contains negligible P. P analyses: mg. P/vessel 'Apparent Easily % conversion inorg. P' hydrol. P of Cori ester Initial 0-02 0-359 Incubated for 1 hr. at 380. Freshly prepared enzyme: Exp. VIII Oxygen 1 atm. 0-018 0-242 33 Nitrogen 1 atm.* 0-017 0-126 65 Freshly prepared purified enzyme is highly susceptible to oxygen. Incubated for 1 hr. at 380. Enzyme had been kept 2 days; centrifuged from precipitate: Exp. IX Oxygen 4-4 atm. 0-016 0-249 31 Nitrogen 1 atm.t 0-018 0-229 36 Exp. X Oxygen 1 atm. 0-02 0-239 33 Nitrogen 1 atm. (triplicate) 0-02 0.220* 39 0-02 0-226t 37 0-02 0-247t 31 Now almost completely insensitive to oxygen again. * Nitrogen purified by hydrosulphite and 'silver-salt'. t Unpurified cylinder nitrogen. $ Nitrogen purified by alkaline pyrogallol. 184 F. DICKENS I946 tivities in oxygen and nitrogen. Although the cinoxidase by quinones, already much studied (e.g. accelerating effects of traces of metals (manganese, Quastel, 1933; Potter, 1942). The statement of cobalt, magnesium) and the inhibitory effects of Engelhardt & Sakov that phosphoglucomutase is other metals (copper, zinc) were fully confirmed, unaffected by oxidizing agents is understandable the presence of these metals did not alone bring in view of the great difference in oxygen sensitivity back the oxygen-sensitivity to the enzyme prepara- of different preparations of this enzyme described tion. The evidence makes it probable that the above. protein precipitate, mentioned above, which was Of these five enzymes, four at least are considered formed on keeping the phosphoglutomutase in the to rely upon intact sulphydryl groups within the cold room, was concerned in the sensitization to enzyme for their activity (enzyme 1, Gill & Leh- oxygen. It is of interest to note that Cori et al. mann, 1939; Lehmann, 1939: enzyme 3, Rapkine, (1938) observed that a similar kind of precipitate, 1938; Rapkine & Trpinac, 1939: enzyme 4, Hopkins added together with magnesium ions, greatly in- & Morgan, 1938; Hopkins, Morgan & Lutwak-Mann, creased the catalytic of these ions on enzyme 1938; Morgan & Friedmann, 1938: enzyme 5, activity. Heating to 700 caused this precipitate to' Barron, 1936; Peters, 1940; Barron & Singer, 1943, lose this co-activating action, and Cori et al. (1938) 1945). The -SH nature of enzyme 2, phospho- consider that the precipitate contained 'an unknown hexokinase, is made highly probable by the work component' of the complete enzyme system. From ofEngelhardt & Sakov (1943). Taken in conjunction our results it appears likely that this, or a similar, with the evidence of poisoning by oxygen of these component may be needed to sensitize phospho- enzymes, these observations make clear a fairly glucomutase to oxygen. Possibly such a component general connexion between the oxygen poisoning may play an important part in the behaviour and the -SH nature of the enzymes involved in towards oxygen of other enzymes: it may very carbohydrate metabolism. This agrees with the well be itself an -SH protein. known oxygen-sensitivity of some other -SH enzymes not concerned in carbohydrate metabolism (cf. Table 1). DISCUSSION Of these five enzymes, the pyruvate oxidase and The evidence collected shows that at least five of the succinic oxidase systems are believed to require the enzymes believed to be concerned in the carbo- in addition to the -SH component a flavoprotein. hydrate metabolism of brain tissue are capable of Evidence of the presence of a flavoprotein in being inhibited by oxygen. These are: (1) phospho- alkali-inactivated succinoxidase has been provided glucomutase, (2) fructose-6-phosphate phosphory- by Straub (1941), but apparently this flavoprotein lase, (3) triosephosphate,dehydrogenase, (4) succinic is not concerned in succinate metabolism directly, dehydrogenase, (5) pyruvic oxidase. In addition since Straub considered that it did not react with cytochrome oxidase may suffer slight damage after succinic dehydrogenase, but had a direct conhiexion long exposure to oxygen. Zymohexase of yeast with the oxidizing system, i.e. it was presumably may be oxygen sensitive (Warburg & Christian, a cytochrome c reductase capable of transporting 1942), but the same enzyme from animal tissues is hydrogen atoms from reduced cozymase. Potter more stable (cf. Table 1).* & Albaum (1943) discuss the possibility that in- In the present paper, and the preceding one activation of such a flavoprotein might indirectly (Dickens, 1946), the oxygen sensitivity of enzymes, inhibit the succinic oxidase system. However this 1, 3, 4 and 5 has been demonstrated by their may be, it does not seem likely from Straub's inhibition by molecular oxygen. That of enzyme 2 experiments that the flavoprotein could be very (phosphohexokinase), which was not investigated labile, since it withstood an hour's exposure to here, is presumed from the work of Engelhardt & pH 9, the conditions used to inactivate the dehydro- Sakov (1943), available only in abstract form; it is genase. So little is known, however, ofthe properties not clear from the abstract whether this inactivation of the flavoprotein component of succinoxidase that occurs only under artificial conditions, viz. by there remains a possibility that it might be oxygen- reason of the quinone formed from added polyphenol sensitive, although experimental evidence of this is by succinoxidase preparations, in which case it lacking. There is a similar ignorance about the would resemble the inactivation of urease and suc- stability to oxygen of the flavoprotein component of the pyruvate oxidase system. * Preliminary reports (Colowick & Price, 1945a, 1945b) The evidence obtained in the present work with indicate that in muscle extracts dihydrocozymase and other flavoproteins (diaphorase, d-amino-acid oxi- {uanine may be essential coenzymes for the phosphorylation dase) and with flavine-adenine-dinucleotide ofbrain, of glucose or fructose-6-phosphate. Although with yeast to of hexokinase no sensitivity to oxygen was observed in the has not demonstrated any sensitivity oxygen present work, these new muscle systems involving dihydro- these substances. On the whole there is, as yet, cozymase would be expected to be oxygen-sensitive. very little positive evidence which would involve VoI. 40 OXYGEN POISONING OF ENZYMES 185 the flavoproteins in the oxygen poisoning of animal how the -SH group partakes in the activity of the tissues. enzyme; not by alternate oxidation and reduction, The evidence involving the -SH enzymes, on apparently, since the inactive form of the enzyme is the contrary, is quite strong. In addition to the not more readily reactivated in presence of succi- known oxygen-sensitivity, further support is de- nate, rather the reverse (Hopkins et al. 1938; see, rived from consideration of the mode of protection however, Potter & Du Bois, 1943). against oxygen-poisoning by metals, inhibitors, and It seems desirable to consider how the presence prosthetic groups. of an activating metal in the succinic dehydrogenase The succinate system, though not that mainly system would affect the interpretation of these affected by oxygen in animal tissues (Mann & results, though it is not suggested that the evidence Quastel, 1946; Dickens, 1946), is nevertheless the available is sufficient to justify such an assumption. clearest example for discussion, because the funda- The fact, however, that the activity of the dehydro- mental work of Hopkins & Morgan (1938) and genase is protected from inactivation by oxygen Hopkins et al. (1938) has made it the best under- in presence of traces of manganese makes the par- stood. Besides revealing for the first time the ticipation of an activating metal at least a possi- essential -SH nature of the dehydrogenase, these bility. This metal need not be manganese; alu- authors showed that the oxidative inactivation of minium, for example, has also been considered to the enzyme by oxidized glutathione was prevented protect this enzyme against reagents which combine by malonate and, rather less effectively, by pyro- with -SH groups (Schneider & Potter, 1943). phosphate. These reagents did not, however, prevent Chromium and rare earth metals also activate the reactivation of the inhibited (oxidized) form succinic oxidase, purified preparations of which con- of the enzyme. It is usually considered (Leloir tain aluminium and chromium (Horecker, Stotz & & Dixon, 1937) that these two inhibitors attach Hogness, 1939). The observations of Massart (1939) themselves to the active centres normally occupied are quite inadequate to support his claim that by the substrate, which would account for the manganese is a component. We have shown, how- specificity of their action; Krebs (1943) however ever (Dickens, 1946; and present work) that cobalt, has stated that pyrophosphate, unlike malonate, an activator of bacterial pyruvic oxidase, protects does not cause any accumulation of succinate in animal pyruvic oxidase against oxygen poisoning, metabolizing tissue, suggesting that these two while the whole glucose-oxidizing system of brain poisons may have qualitatively different affinities. is similarly protected by magnesium, manganese Adler, Euler, Gunther & Plass (1939) consider, but or cobalt. Consequently much more extensive reject, the possibility that pyrophosphate may experiments than those recorded in this paper, poison an essential metal component (manganese which were designed with quite a different end in or magnesium) of succinoxidase. view, are highly desirable to see if further evidence As regards the inactivation of succinic dehydro- of a metal component of the succinic system can genase by oxygen, the experiments described here, be obtained. Such a metal could be a link between like those obtained independently by Stadie (1943), the -SH groups and the substrate (cf. Hellerman, show clearly that the presence of malonate during 1937) or inhibitor, which would offer a possible the exposure to oxygen protects this enzyme, just explanation of the effect of inhibitors in holding as it is protected by malonate against inactivation the enzyme in the reduced form and thus protecting by exposure to oxidized glutathione (Hopkins & it against oxidants, while they do not prevent the Morgan, 1938) or benzoquinone (Potter & DuBois, reduction of the S-S form of the enzyme. There are 1943), and it is presumed that the same mechanism difficulties in the way of this theory, such as the is involved. (In a single experiment, however, we problem of what happens to the metal when the observed no protection of heart muscle succinoxi- enzyme is in the oxidized form, but nevertheless dase against high-pressure oxygen by pyrophos- it appears to merit some consideration. phate; this should be repeated as, ifit were confirmed, It is necessary to point out that malonate does it might indicate a different mechanism of com- not exert its protective influence by virtue of the bination of pyrophosphate from that of malonate.) inhibition of enzyme activity which it produces. It is not yet possible to give more than a general Activators can also protect this and other -SH picture of the manner of malonate protection. enzymes, as we have seen. Similarly, it has been Malonate appears not to react with -SH groups, shown above that cozymase protects its enzyme, and it is necessary to assume that the latter are triosephosphate dehydrogenase, against inactiva- 'shielded' by the steric effects of the malonate tion by oxygen, and Rapkine (1938) and Rapkine molecules which are supposedly adsorbed at closely & Trpinac (1939) showed a similar protection against adjacent positions on the surface of the enzyme the usual chemical oxidants. The metals which pro- (cf. Potter & Du Bois, 1943), chemically perhaps tect pyruvic oxidase have already been discussed. not a very satisfying explanation. Nor is it known The protection of apoenzymes by their prosthetic 186 F. DICKENS I946 groups or substrates is a commonplace of enzyme 3. Lactic and malic dehydrogenase preparations chemistry. All these substances have one feature from animal tissues were very resistant to oxygen in common; they are specifically adsorbed at (or inactivation. possibly adjacent to) the active centres of the 4. Triosephosphate dehydrogenase is poisoned dehydrogenases which they protect. Not every such by oxygen, but is sensitive only when the exposure substance, however, is necessarily protective. Zymo- occurs in absence of cozymase, the coenzyme of hexase of yeast forms 'salts' with cobalt and iron this dehydrogenase. which, in presence of cysteine, are reversibly in- 5. Typical flavoproteins (d-amino-acid oxidase, activated by molecular oxygen, but the zinc com- diaphorase) were not affected by exposure to pound of the same enzyme is not inactivated by 4-4 atm. ofoxygen. The flavine-adenine-dinucleotide oxygen (Warburg & Christian, 1942, 1943). Conse- content of brain tissue was not affected by oxygen quently the nature of the activating metal and its at 3-5 atm. mode of combination, as well as the firmness with 6. Catalase in brain tissue is insensitive to which it is bound, and the nature of the -SH oxygen. 'contaminants', determine whether the enzyme is 7. Choline oxidase, an -SH enzyme, is poisoned sensitive to oxygen or not. The great complexity by oxygen. introduced by these factors in studies of enzyme 8. The activity of yeast hexokinase was not inactivation by oxygen is evident. They may explain affected by oxygen (glucose present). the many confusing differences in the stability to 9. Phosphoglucomutase is poisoned by oxygen, oxygen of various preparations of the same enzyme, but only at certain stages of its purification. which have been repeatedly seen during the course 10. The relationship between oxygen-sensitivity of the present work and also by many other ob- and the presence of essential -SH groups in many servers. of the sensitive enzymes is pointed out. The possible SUMMARY mechanism of inactivation and of protection is dis- 1. A list has been compiled of enzymes known cussed, and suggestions are advanced concerning or considered to be oxygen-sensitive. the probably important part played by trace-metals 2. Succinic oxidase is inhibited by exposure to in these processes. high oxygen tensions. The dehydrogenase, the most sensitive component, is apparently irreversibly This work, like that in the preceding paper, was done poisoned. Longer exposure may also somewhat between March 1943 and March 1944 at the request of weaken the cytochrome oxidase. Evidence is pre- the Royal Naval Personnel Research Committee of the sented that a minor part of the Medical Research Council. I am most grateful to the probably only Council for giving me this opportunity of studying oxygen poisoning could be accounted for by accumula- poisoning, and for the permission now granted to publish tion of oxaloacetate in oxygen. Malonate (103M) these results. Dr William C. Stadie has courteously given or manganese ions (2.5 x 104M) protect the de- me permission to refer to some unpublished observations hydrogenase against poisoning by oxygen. by himself and his colleagues.

REFERENCES Adler, E., Euler, H. v., Gunther, G. & Plass, M. (1939). Colowick, S. P. & Price, W. H. (1945b). J. biol. Chem. 159, Biochem. J. 33, 1028. 563. Axelrod, A. E., Potter, V. R. & Elvehjem, C. A. (1942). Colowick, S. P. & Sutherland, E. WV. (1942). J. biol. Chem. J. biol. Chem. 142, 85. 144, 423. Bailey, B., Belfer, S., Eder, H. & Bradley, H. C. (1942). Cori, G. T., Colowick, S. P. & Cori, C. F. (1938). J. biol. J. biol. Chem. 143, 721. Chem. 124, 543. Barron, E. S. G. (1936). J. biol. Chem. 113, 695. Corran, H. S., Green, D. E. & Straub, F. B. (1939). Biochem. Barron, E. S. G. (1943). Advances in Enzymology, 3, 149. J. 33, 793. New York: Interscience Publ. Inc. Dickens, F. (1946). Biochem. J. 40, 145. Barron, E. S. G. & Singer, T. P. (1943). Science, 97, 356. Dixon, M. (1925). Biochem. J. 19, 507. Barron, E. S. G. & Singer, T. P. (1945). J. biol. Chem. 157, Dixon, M. (1940). Personal communication. 221. Dixon, M. & Kodama, K. (1926). Biochem. J. 20, 1104 Bohr, D. F. & Bean, J. W. (1940-1). Amer. J. Physiol. 131, (p. 1109). 388. Edlbacher, S., Kraus, J. & Leutdhardt, F. (1933). Hoppe- Cohen, P. P., Hekhius, G. L. & Sober, E. K. (1942). Cancer Seyl. Z. 217, 85. Res. 2, 405. Edson, N. L. (1935). Biochem. J. 29, 2082. Colowjck, S. P. & Kalekar, H. M. (1941). J. biol. Chem. 137, Engelhardt, V. A. & Sakov, N. E. (1943). Biochimia, 8, 9 789. (Brit. Chem. and Phy8iol. Ab8tr. 1944, A in, 367). Colowick, S. P. & Price, W. H. (1945a). J. biol. Chemn. 157, Fujita, A. & Kodama, T. (1931). Biochem. Z. 232, 20. 415. Gale, E. F. (1939). Biochem. J. 33, 1012. Vol. 40 OXYGEN POISONING OF ENZYMES 187 Gill, P. M. & Lehmann, H. (1939). Biochem. J. 33, 1151. Negelein, E. & Bromel, H. (1939). Biochem. Z. 300, 225. Green, D. E., Needham, D. M. & Dewan, J. G. (1937). Ochoa, S. & Rossiter, R. J. (1939). Biochem. J. 33, 2008. Biochem. J. 31, 2327. Perlzweig, W. A. (1932). Science, 76, 435. Haas, E., Horecker, B. L. & Hogness, T. R. (1940). J. biol. Peters, R. A. (1940). Personal communication. Chem. 136, 747 (p. 773). Potter, V. R. (1942). Cancer Res. 2, 688. Hanes, C. S. (1940). Proc. Roy. Soc. B, 129, 174. Potter, V. R. & Albaum, H. G. (1943). J. gen. Physiol. 26, Hellerman, L. (1937). Physiol. Rev. 17, 454. 443. Hellerman, L., Perkins, M. E. & Clark, WV. NI. (1933). Proc. Potter, V. R. & Du Bois, K. P. (1943). J. gen. Physiol. 26, Nat. Acad. Sci., Wash., 19, 855. 391. Herbert, D., Gordan, H., Subrahmanyan, V. & Green, D. E. Quastel, J. H. (1933). Biochem. J. 27, 1116. (1940). Biochem. J. 34, 1108. Quastel, J. H. & Wheatley, A. H. M. (1938). Biochem. J. Hobermann, H. D. & Rittenberg, D. (1943). J. biol. Chem. 32,. 936. 147, 211. Rapkine, L. (1938). Biochem. J. 32, 1729. Hopkins, F. G. & Morgan, E. J. (1938). Biochem. J. 32, 611. Rapkine, L. & Trpinac, P. (1939). C.R. Soc. Biol., Paris, Hopkins, F. G., Morgan, E. J. & Lutwak-Mann, C. (1938). 130, 1516. Biochem. J. 32, 1829. Richards, M. M. & Hellerman, L. (1940). J. biol. Chem. 134, Horecker, B. L., Stotz, E. & Hogness, T. R. (1939). J. biol. 237. Chem. 128, 251. Schneider, W. C. & Potter, V. R. (1943). J. biol. Chem. 149, Irving, G. W., Fruton, J. S. & Bergmann, 3I. (1942). J. biol. 217. Chem. 144, 161. Shapiro, B. & Wertheimer, E. (1943). Biochem. J. 37, 102. Kalekar, H. M. (1943). J. biol. Chem. 148, 127. Sizer, I. W. (1942). J. biol. Chem. 145, 405. Klein, G. & Zeise, W. (1933). Hoppe-Seyl. Z. 222, 187. Stadie, W. C. (1943). Personal communication. Klein, G. & Zeise, WV. (1935). Klin. Wschr. 14, 205. Stadie, W. C., Riggs, B. C. & Haugaard, N. (1944). Amer. Krebs, H. A. (1943). Advances in Enzymology, 3, 191. J. Med. Sci. 207, 84. New York: Interscience Publ. Inc. Stephenson, M. & Stickland, H. (1931). Biochem. J. 25, Lehmann, H. (1939). Biochem. J. 33, 1240. 205. Lehmann, J. (1935). Skand. Arch. Physiol. 72, 78. Stern, K. G. & Melnick, J. L. (1941). J. biol. Chem. 139, 301. Leloir, L. F. & Dixon, NI. (1937). Enzymologia, 2, 81. Straub, F. B. (1939). Biochem. J. 33, 787. Liebricht, W. & Massart, L. (1937). C.R. Soc. Biol., Paris, Straub, F. B. (1941). Enzymologia, 9, 148 (cited from 124, 299. Potter & Albaum, 1943). Mann, P. J. G. & Quastel, J. H. (1937). Biochem. J. 31, 869. Sumner, J. B. & Poland, L. 0. (1933). Proc. Soc. Exp. Biol., Mann, P. J. G. & Quastel, J. H. (1946). Biochem. J. 40, 139. N.Y., 30, 553. Mann, P. J. G., Woodward, H. E. & Quastel, J. H. (1938). Swingle, K. F., Axelrod, A. E. & Elvehjem, C. A. (1942). Biochem. J. 32, 1024. J. biol. Chem. 145, 581. Marks, G. WV. (1936). J. biol. Chemn. 115, 299. Warburg, 0. (1926). Biochem. Z. 172, 432. Marks, H. P. (1944). Report to Royal Naval Personnel Warburg, 0. & Christian, W. (1938). Biochem. Z. 298, 150. Research Committee, Med. Res. Coun. no. 101. Warburg, 0. & Christian, W. (1942). Biochem. Z. 311, 209. Massart, L. (1939). Hoppe-Seyl. Z. 258, 190. Warburg, 0. & Christian, W. (1943). Biochem. Z. 314, 149. Meyerhof, 0. (1927). Biochem. Z. 183, 178. Williamson, S. & Green, D. E. (1940). J. biol. Chem. 135, Morgan, E. J. & Friedmann, E. (1938). Biochemii. J. 32, 862. 345.