sign in sign up
<<
Home , Acetaldehyde, Alcohols (medicine)

914 NATURE June 12, 1948 Vol. 161 These are valid equivalents ; but the important point is this-the agreed figure for the last two THE -SUBSTRATE COM• whaling seasons of 16,000 'blue whale units' for the POUNDS OF AND contracting parties may well mean that a full catch involves the killing of many more than 16,000 PEROXIDES animals. For example, in 1945-46 the Antarctic catch was returned8 as 8,304·8 'blue whale units', By BRITTON CHANCE* but was, in fact, made up of 13,381 whales (3,604 blue, 9,184 fin, 238 humpback and 81 sei whales, as well Biochemical Department, Medical Nobel Institute, Stockholm, and Molteno Institute, University of as 239 sperm whales and 35 "sperm and others"). Cambridge This last might include some bottlenose or Berardius. The catch for 1946-47 has been returned7 as 15,230·7 'blue whale units'. This represents 8,870 blue, ATALASE is a hrematin enzyme found in 12,857 fin and 2 sei whales, a total of 21,729 C relatively large concentrations in erythrocytes, whales8 • , kidney, etc. Its extremely efficient catalysis There is obviously a serious danger of losing sight of the destruction of peroxide and its of the reality of the position by continued contempla• ability to catalyse the oxidation of by tion of the 'blue whale unit' value of a catch, since have been recognized for some the number of animals killed will always be greater time, but very little direct evidence for the than the number of units allowed. An industry of mechanisms of these reactions in terms of the this sort cannot be regulated by reference to abstrac• theory of enzyme-substrate compounds has hitherto tions, when it depends entirely upon a stock of wild been obtained. animals, over the and breeding habits of which Using a micro form of the method of Hartridge there is no control. and Roughton1 and Millikan2 for rapid spectrophoto• In the presence of vast and unestimated numbers metric studies3 of the reaction kinetics and equilibria of animals which it is desired to exploit commercially, of the unstable compounds of catalase with hydrogen nothing is easier than to persuade oneself that the peroxide and methyl or ethyl hydrogen peroxide, the stock is so great that it is impossible to injure it, still catalytically active enzyme-substrate compounds of less to destroy it. But it is my opinion that the stocks catalase with these substances have been discovered. of blue whales are showing clear signs of reduction : In addition, corresponding inactive forms of the (1) in the rarity or absence of the huge animals of compounds of catalase with hydrogen peroxide and earlier seasons ; (2) in the reduced proportion in the methyl hydrogen peroxide have been revealed. The total catch of this species ; and (3) in the reduced catalase-ethyl hydrogen peroxide complex which had percentage of mature females which were pregnant. previously been studied by Stern4 is shown to be one It is also certain that a parallel reduction of the stock of these inactive enzyme-substrate compounds. The of fin whales is now in progress. three active enzyme-substrate complexes share a The position with regard to sperm whales is some• common property of oxidizing substances such as what different. These animals are polygamous, and alcohols, and in this case catalase functions as a there are therefore spare bulls which can very well peroxidase. But the active catalase- hydrogen per• be utilized. The sperm whales occurring outside the oxide complex has the unique property of acting warm of the breeding grounds are almost 'catalatically', that is, of decomposing hydrogen invariably large males, which have not been able to peroxide directly into and . These keep their place in the herds. There is, however, no studies have shed some light on the mechanisms of information as to the size of this stock of spare bulls, these reactions. or whether they are resident in the Antarctic. Hence, the present increased killing may simply be using up Spectra of the Primary and Secondary the accumulation of years, either of a resident male stock, or of an annually visitant herd. The appro• Compounds priate literature is not available here; but I have the clearest recollection that in the days of the old sperm The colour of the three active or primary (I) whalers, certain solitary bulls were known to haunt enzyme-substrate compounds of catalase is pale particular areas of the sea, and continued to do so green, and they have an absorption band at about for years. Such whales, like the authentic 'Moby 670 m:.L. The Soret band is decreased in intensity Dick' and 'Paita Tom', attacked boats at sight. and is slightly shifted towards the visible region of the spectrum, giving an isobestic point at about Finally, it may not be out of place to recall to 5 memory the fate of the North American bison and 435 miJ. • At 405 miJ. the decrease of extinction the passenger pigeon. The former was brought to coefficient per mole of hrematin iron bound by the verge of extinction and the latter completely peroxide is the same for the three primary com• exterminated by man for commercial purposes, in plexes. Under suitable conditions the green com• spite of the almost incredible numbers in which they pounds change into inactive or secondary (II) red both existed a little more than a century ago. compounds having two bands, at 536 and 572 miJ., approximately in the positions found by Stern4 for 1 N Hvalfangst Tidende, No. 11, 278 (1946). catalase-ethyl hydrogen peroxide. The green-red 'Mackintosh N. A., and Wheeler, J. F. G., "Southern Blue and Fin shift of the hydrogen peroxide compounds super• Whales' 1, Discovery Reports, 1, 417 (1929). 'Mackintosh, N. A., "The Southern Stock of Whalebone Whales", ficially resembles that found by Theorelle for horse• Discovery Reports, 22, 217 (1942). radish peroxidase and hydrogen peroxide. By 1 Matthews, L. H., "The Humpback ·whale", Discovery Reports, 17, 93 (1937). analogy with his views, the green primary compounds ' L mrie, A. H., "The Age of Female Blue Whales", Discovery Reports, are ferric iron peroxide complexes with ionic bonds, 15, 265 (1937). and the red secondary compounds are ferric iron 1 Norst Hvalfangst Tidende, No. 11, 277 (1946). peroxides with covalent bonds. 'Norst Hvalfangst Tidende, No. 3, 83 (1947). • N orst Hvalfangst Tidende, No. 9, 322 (1947). • John Simon Guggenheim Memorial Fellow.

©1948 Nature Publishing Group No. 4102 June 12, 1948 NATURE 915 Molecular Composition of Catalase- Hydrogen tration of about IQ-9 M hydrogen peroxide is required Peroxide for half-saturation of the primary catalase- hydrogen peroxide complex). This high affinity is not obtained In the primary compound of erythrocyte catalase when hydrogen peroxide 'from the bottle' is added to and hydrogen peroxide three out of four hrematin• catalase, for then the value is 1·6 X 10-e M (at a iron groups of catalase can still react with 3·4 x 1o-• M catalase concentration&) due to the ion or alkyl hydrogen peroxide. Thus the maximum p.:trtial destruction of hydrogen peroxide concentration of catalase-hydrogen peroxide found formation of the primary catalase -hydrogen peroxide under any condition corresponds to only one of the complex. Catalase has an affinity equivalent to total number of hrematins present in the catalase about l()-8 M for methyl hydrogen peroxide and . But the alkyl hydrogen peroxides combine about 10--t M for ethyl hydrogen peroxide. These with all four hrematins, as is shown by the inability values are all increased in the presence of substances of their catalase compounds to combine with cyanide. such as alcohols which cause the decomposition of This difference in composition of the primary hydro• these complexes. gen peroxide and alkyl hydrogen peroxide compounds The secondary compounds of catalase and these causes a four-fold difference in the decrease of the peroxides are formed from the primary compounds intensity of the Soret band of catalase when these comparatively slowly, are much more stable than the compounds are formed. primary compounds, and do not . t? react While a catalase in which all four hrematin groups directly with alcohols. They are thus inhibitors of are combined with alkyl hydrogen peroxide is the catalytic activity of the primary complexes. relatively stable except in the presence of alcohols, the attachment of more than one molecule of hydro• Reactions of the Primary Complexes gen peroxide to catalase results in an as yet un• with Alcohols, etc. detected complex which rapidly decomposes. It is Keilin and Hartree&,10 have demonstrated that this 'catalatic' activity which limits the saturation catalase and hydrogen peroxide or ethyl hydrogen value of the primary catalase-hydrogen peroxide peroxide can oxidize alcohols. These studies show complex to one hydrogen peroxide molecule per that the primary catalase peroxide complexes are the catalase molecule. This complex is the enzyme• active agent in these oxidations and are true Michaelis substrate compound concerned in the decom• compounds since they fulfil the tests applied to the position of hydrogen peroxide, but it does not peroxidase-hydrogen peroxide The re• exhibit the properties of a Michaelis intermediate action-velocity constants for the reactiOn of the compound because the hydrogen peroxide not only primary compounds with alcohols, etc. (calculated unites with catalase to form the enzyme-substrate from the formulre developed for peroxidase8 ), are very complex but also reacts again with this complex to nearly independent of whether hydrogen peroxide or cause the decomposition of hydrogen peroxide. methyl or ethyl hydrogen peroxide is bound to Therefore, no Michaelis saturation effect is to be catalase, and have values which decrease with the expected, and the velocity of destruction of hydrogen increasing size of the molecule, as is the case peroxide by strong catalase solutions is directly pro• with the increasing size of the peroxide molecule. portional to the hydrogen peroxide concentration, at The velocity constant with or is 1 least up to 0 · 3 M • The previous evidence for a k, ..-...; 1000 M-1 sec.-1 (the turnover of the primary Michaelis constant of 0·025 M appears to be an compounds in the presence of 0·001 M ethanol is artefact due to the inactivation of catalase. 1000 x 0·001 times per sec.). The reactions of these three catalase peroxides with alcohols are not Kinetics of Formation and Decomposition inhibited by . and the Enzyme-substrate Affinity Direct spectroscopic measurements of the primary Of the three primary complexes, that with hydrogen compound of catalase and hydrogen peroxide have been obtained in the presence of notatin, glucose, peroxide forms the most rapidly (k1 ..-...; 3 X 101 M-1 sec.-1 (see ref. 5), that is, in a 1 M hydrogen peroxide and oxygen. The absolute spectrum of the compound solution the complex would be half-formed in about has been obtained with the Beckman spectrophoto• meter in the region mfJ. and completely con• 2·3log2 . X sec.) and the larger the peroxide molecule, firms the spectrum obtained using 'bottle' hydrogen 3 101 peroxide and the rapid flow technique&. the slower the reaction (..-...;I X 108 and ..-...; 1 X 10' The direct spectroscopic studies of the turnover M.-1 sec.-1 for methyl and ethyl hydrogen peroxide number of the catalase peroxides in presence of respectively). This decrease is much greater than different alcohols agree with Keilin and Hartree's could be expected solely upon the basis of diffusion data obtained in coupled oxidations10. In such velocities and must be attributed mainly to a steric coupled oxidations the saturation of the catalase• effect, due possibly to restricted accessibility of hydrogen peroxide complex is found to be small in catalase hrematin to these substances. In these three spite of the extremely high affinity of catalase for reactions, the kinetics of the formation of the primary hydrogen peroxide. Therefore, the steady state complexes clearly represent the direct combination hydrogen peroxide concentration during efficient of catalase with peroxide without the formation of coupled oxidations is only about 1()-' M. Only under any detectable intermediates. The primary com• these conditions is all the hydrogen peroxide utilized plexes are not stable ; they decompose spontaneously for and no molecular oxygen is into free catalase with a velocity constant of evolved. about 0·02 sec.-1 (the half-time for the reaction is Two other biologically important substances have 2·3 log 2 been found to react with catalase peroxides just as . sec.) even in the complete absence of 0 02 rapidly as do ethanol or methanol. These are form• alcohols. (probably as glycol) and formate. Catalase shows the highest affinity for hydrogen By a combination of the chromotrophic acid method11 peroxide 1o-• M (that is, a steady-state concen- for production or disappearance and

©1948 Nature Publishing Group 916 NATURE June 12, 1948 Vol. lbl an acidimetric method using brom-cresol purple for secondary complex ensues, and the two-banded production and disappearance, the con• spectrum (536 and 572 f.l) may be seen in the hand version of methanol to formaldehyde and formic acid, spectroscope. Since these bands are close to those and the destruction of the latter, have been determ• reported by Lemberg and Foulkesu for a compound ined using ethyl hydrogen peroxide as the sub• of catalase and ascorbic acid, and since hydrogen strate. Similarly, production from peroxide formed by autoxidation of ascorbic acid has formate can be demonstrated in 'coupled oxidations' already been shown to give rise to the primary com• in which hydrogen peroxide is the substrate (Keilin plex•, there appears to be little evidence for the and Hartree, personal communication). existence of this catalase-ascorbic acid complex. The reaction of catalase-ethyl hydrogen peroxide The secondary complex has not yet been observed with ethanol apparently gives acetaldehyde from spectroscopically by adding 'bottle' hydrogen per• ethanol and ethanol from ethyl hydrogen peroxide ; oxide to catalase owing to the decomposition of the the supply of ethanol is not at all depleted during hydrogen peroxide in a fraction of a second. Under the oxidation, and a small initial ethanol concen• the conditions used in the determination of Kat F, tration may cause the reduction of a large amount of catalase is in contact with 'bottle' hydrogen peroxide ethyl hydrogen peroxide. The destruction of strong for ten minutes or more. The observed slow decrease ethyl hydrogen peroxide solutions by catalase studied of activity during this time has been proved to be by Stern•, in which catalase is almost completely due mainly to the formation of the secondary com• converted into the secondary compound, can be plex, since after the secondary complex has been explained on the basis of a small percentage of the formed by notatin, glucose, and oxygen, the diluted primary compound in equilibrium with the secondary solution has only half the Kat F of the untreated compound. This primary compound reacts rapidly solution. Furthermore, at pH 3·5 the rate of decrease with ethanol present in ethyl hydrogen peroxide, and of activity7 and the rate of formation of the secondary a small initial ethanol concentration will therefore complex in the presence of notatin, glucose, and cause the reduction of all the ethyl hydrogen per• oxygen are both accelerated. The extent of in• oxide. Thus the normal reactions of the primary activation of catalase depends upon the number of compound were masked by the presence of Stern's catalase hrematins converted to the secondary com• inactive red complex. plex, and this varies with the experimental con• The fact that catalase has a high affinity for ethyl ditions. hydrogen peroxide appears to contradict the results This explanation of the inactivation of catalase by obtained by Stern13, who failed to find appreciable hydrogen peroxide justifies the new method for the inhibition of the destruction of hydrogen peroxide by determination of catalase activity, in which strong catalase until about 0 ·1 M ethyl hydrogen peroxide enzyme solutions are used so that the reaction is solutions were used. However, these spectroscopic complete in a few minutes7• In this procedure there measurements show the 'stripping off' of alkyl is insufficient time for the formation of the secondary hydrogen peroxide groups bound to catalase hrematin complex and a nearly constant activity is therefore upon the addition of hydrogen peroxide, with the observed. result that only the catalase-hydrogen peroxide com• plex remains. In experiments in which hydrogen Reaction Mechanisms peroxide is continuously generated by notatin, glucose and oxygen, the rapid turnover of a large The nature of the reaction velocity constants amount of catalase-bound alkyl hydrogen peroxide obtained in these experiments suggests the following is observed spectroscopically. Thus hydrogen per• overall equations. The symbol "Cat" represents the oxide can take the place of an alcohol and can react four ferric-iron atoms of catalase hrematin to which with catalase alkyl peroxides. It does so with about the peroxides become attached. The hydroxyl group the same velocity as it reacts directly with catalase. found by Agner and TheoreUU is considered to be Therefore hydrogen peroxide has a reactivity of replaced by the peroxide group. The equations are . (3 X107) written in terms of methyl hydrogen peroxide and about 30,000 times 103 that of ethanol. ethanol for simplicity, but similar reactions have been measured with ethyl hydrogen peroxide as By a comparison of the activity of catalase• substrate, and with several lower alcohols, form• peroxides towards methanol, methylene glycol, and aldehyde, and formate in place of ethanol. formic acid (all three are equally active) and ethanol, acetaldehyde, and (the latter two are nearly 1 x 10' M-1 sec.-' inactive), it is concluded that both a hydroxyl group Cat(OH)4 + 4CH300H 4H20 + and a hydrogen atom attached to the one carbon <0·01 sec.-' Cat(- OOCH ),- OOCH ) - II. (l) atom are required of a suitable oxygen acceptor. 3 3 4 The acceptor action of hydrogen peroxide constitutes an exception to this rule. Since the reaction velocity Cat( - OOCHs), - I + 4C.HsOH uoo M-1 sec.-' of both substrate and acceptor decreases with in• Cat(OH), + 4CH30H + 4CH3CHO. (2) creasing size of the acceptor or substrate molecule, the acceptor molecule may have to pass through the Thus the oxidation of alcohols by the primary same 'hole' as the substrate and may momentarily catalase alkyl hydrogen peroxide complexes follows become attached to or near the catalase iron peroxide the simple theory of Michaelis and Menten, where complex. Cat( -OOCH3 ),-l is the Michaelis compound. The conversion of this primary compound into the Secondary Compound of Catalase and secondary compound is negligible when the alcohol concentration is large and the peroxide concentration Hydrogen Peroxide is small. When catalase is continuously supplied with In Stern's experiments• the ethyl hydrogen per• hydrogen peroxide by means of notatin, glucose, and oxide concentration exceeded the ethanol concen• oxygen, the slow conversion of the primary to the tration, and catalase was mainly in the form of the

©1948 Nature Publishing Group No. 4102 June 12, 1948 NATURE 917 secondary complex which he mistook for the active secutive reactions of hydrogen peroxide with catalase enzyme-substrate complex. and catalase-hydrogen peroxide are involved, and The oxidation of alcohols by the primary catalase the saturation value of the catalase-hydrogen per• hydrogen peroxide complex may also be regarded as oxide complex depends upon the relative velocities following the Michaelis theory if proper allowance is of these reactions. The formation of a catalase made for the reactions of this complex with hydrogen hydrogen peroxide complex containing one hydrogen peroxide (see equation 5) and for the fact that only peroxide molecule per four catalase hrematins can be one of the four catalase hrematins is bound to hydro• explained in two ways : gen peroxide in this complex. A purely 'kinetic' explanation is that the ratio of the velocity constant for the hydrogen peroxide ,.._, 3 x 10' M-•sec.-• reaction of equation 5 to that of equation 3 is 3 : 1 ; Ca.t(OH), + H 20 2 H 20 + <0·02 sec.-• hence the maximum concentration of the catalase• hydrogen peroxide complex is a quarter the enzyme Ca.t(OH) 3 (-OOH)- I*-Cat(OH) 3(00H)- II. (3) concentration. In this mechanism for equation 5, 1000 M-• sec.-• the hydrogen peroxide molecule may collide directly Cat(OH)a(OOH) -I + C2H,OH with the peroxide group attached to hrematin or may Cat(OH), + CH3CHO + H 20. (4) collide with one of the three free catalase hrematins to give, in either case, oxygen, water and free catalase. The overall mechanism for the destruction of In the latter case, a catalase with only one hrematin hydrogen peroxide by catalase is represented: group would be 'catalatically' inactive (for a more detailed discussion see Theorell18). A second explan• ,.._, 3 x 10' M-• sec.-• ation assumes 'special properties' of the catalase• Cat(OH), + H 20 2 hydrogen peroxide complex. The existence of this <0·02 sec.-• Cat(OH) (- OOH)- II. (3) complex is necessary for the decomposition of hydro• 3 gen peroxide into free oxygen and water or into ,.._, 3·5 x 10' M-1 sec.-• radicals at the free catalase hrematins, but the com• Ca.t(OH) 3 (00H) - I + H 20 2 ------plex itself is not decomposed by these reactions. The Cat(OH), + H 20 + o.. (5) complex may, however, decompose spontaneously or on reaction with alcohols, etc. In this case also a Although similar equations were previously pro• one-hrematin catalase would be 'catalatically' in• posed (for a summary see Sumner18), it is only now active. These two mechanisms give very nearly that they find experimental support. First, each step identical mathematical solutions over the ranges of in equation 3 has been observed spectroscopically, experimental conditions that can now be investigated and kinetic data have been obtained. Secondly, the with accuracy, and therefore a final conclusion on the

reactions of Cat(OH)3 (-00H)-I with alcohols exact mechanism for 'catalatic' activity is not yet according to equation 4 have been followed spectro• possible. scopically, as has the following reaction of catalase• A comparison of equations 2, 4 and 6 with equation bound alkyl hydrogen peroxide with hydrogen 5 indicates that there is no fundamental difference peroxide: between the 'peroxidatic' and 'catalatic' reactions ; 3 x 10' M-• sec.-• 'catalatic' activity appears to be the special case of Cat( -OOCHa), + 4H20 2 peroxidatic activity when the substrate and acceptor Cat(OH), + 4CH30H + 402 • (6) are identical. Complete papers on these experiments will be It is therefore concluded that the second step of published elsewhere. 'catalatic' activity is a second-order reaction of I wish to acknowledge my gratitude to those who Cat(OH)8 (-00H)-I with a second molecule of hydrogen peroxide according to equation 5. Thirdly, have taken an active interest in the progress of this equations 3 and 5 indicate that, prior to the formation research and who have made many useful criticisms of appreciable amounts of the secondary complex, and suggestions : Profs. H. Theorell, L. Pauling, D. the overall velocity of decomposition of hydrogen Keilin and F. J. W. Roughton, and Dr. E. F. Hartree. peroxide will increase linearly with initial hydrogen I am indebted to Dr. R. K. Bonnichsen for supplying peroxide concentration over a wide range. This is the bulk of the catalase preparations used in these true at least up to 0·3 M, since the Michaelis constant experiments.

of 0·025 M is an artefact•. In addition, the rapid 1 H ., and Roughton, F. J. W., Proc. Roy. Soc., A, 104, and slow phases of catalase activity found by Georgel7 376 (1923). probably represent catalase activity in the absence • Roughton, F . J. W., and Millikan, G. A., Proc. Roy. Snc., A, 155, 258 (1936). and in the presence of the secondary complex. 'Chance, B., Rei!. Sci. Instr., 68, 601 (1947). In efficient coupled oxidations, the steady-state • Stern, K. G., J . Biol. Chem., 114, 473 (1935). concentration of hydrogen peroxide is so low that the • Chance, B., Acta Chem. Scand., 1, 236 (1947); reactivity velocity constants at 25" C. reaction velocity according to equation 5 is negligible • Theorell, H., Ark. Kemi. Min. o Geol., 16A, No. 3, 1 (1942). compared with that according to equation 4. There• ' Bonnlchsen, R. K., Chance, B., and Theoreli, H., Acta Chem. Scand. fore no molecular oxygen is evolved and hydrogen (in the press). peroxide is utilized quantitatively in the oxidation of • Chance, B., J. Biol. Chem., 151, 553 (1943). alcohol to aldehyde. • Keiiln, D., and Hartree, E. F., Proc. Roy. Soc., B, 119, 141 (1936). 1• Keiiln, D. , and Hartree, E. F., Biochem. J. , 39, 293 (1945). The reactions 1 and 2, 3 and 4, and 1 and 6, repre• 11 MacFayden, D. A. , J. Biol. Chem., 158, 107 (1945). senting the 'peroxidatic' activities of catalase, can be "Rieche, A. , and Hitz., F., Ber., 62, 2458 (1929). analysed according to the Michaelis theory; but " Stern, K. G., Z . physiol. Chem., 209, 176 (1932). equations 3 and 5, representing the 'catalatic' "Lemberg, R. , and Foulkes, E. C., Nature, 161, 131 (1948). destruction of hydrogen peroxide, cannot, since con- "Agner, K., and Theorell, H ., Arch. Biochem. , 10, 321 (1946). "Sumner, J. B., "Adv. in Enz.",1, 170 (1941). • The kinetics of this reaction do not follow first or secon

©1948 Nature Publishing Group