Nature the Enzyme-Substrate Com- Pounds of Catalase and Peroxides

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 ENZYME-SUBSTRATE COM whaling seasons of 16,000 'blue whale units' for the POUNDS OF CATALASE 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 • liver, kidney, etc. Its extremely efficient catalysis There is obviously a serious danger of losing sight of the destruction of hydrogen peroxide and its of the reality of the position by continued contempla ability to catalyse the oxidation of alcohols by tion of the 'blue whale unit' value of a catch, since hydrogen peroxide 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 lives 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 waters of the breeding grounds are almost 'catalatically', that is, of decomposing hydrogen invariably large males, which have not been able to peroxide directly into water and oxygen. 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 cyanide 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 molecule. 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 alcohol molecule, as is the case peroxide by strong catalase solutions is directly pro with the increasing size of the peroxide molecule.

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