618 LIGHT AND LIFE 



acetaklehyde. It indophenol dye were acting by catalyzing the reduc- 

 tion of oxygen to hydrogen peroxide, it should be possible, on the ad- 

 dition of ethanol to the reaction mixture, to observe net oxygen 

 consumption and acetaklehyde production at a rate approaching that 

 of the oxygen exchange observed Avith isotopes, and therefore com- 

 mensurate with the rate of ATP synthesis. 



A necessary preliminary to quantitative comparisons of the Mehler 

 reaction with indophenol-induced oxygen exchange was to test the 

 efficiency of the catalase-ethanol trap for hydrogen peroxide measure- 

 ments. Keilin and Hartree, in first describing the peroxidative ac- 

 tivity of catalase, found that this enzyme could use up to eighty per 

 cent of the available hydrogen peroxide for the peroxidation of 

 ethanol provided the hydrogen peroxide was generated in the reac- 

 tion mixture (4) . However, the conditions for their experiments 

 were quite different from those employed for photophosphorylation, 

 so it seemed imperative to check the efficiency of the catalase-ethanol 

 trap for hydrogen peroxide under these new circumstances. The oxi- 

 dation of alanine by D-amino acid oxidase was used as a hydrogen- 

 peroxide-generating system. In the presence of catalase-ethanol, oxygen 

 consumption by this reaction should be equivalent to the amount of 

 acetaklehyde produced. The reaction Avas carried out in Warburg 

 manometer vessels at fifteen degrees in the presence of all the usual 

 components of the oxidative photophosphorylation reaction mixture. 

 The reaction was stopped by the addition of trichloroacetic acid, the 

 protein removed by centrifugation and the pU adjusted to neutrality. 

 An aliquot of the supernatant fiuid was assayed for acetaklehyde with 

 alcohol dehydrogenase and DPNH (7) . A similar aliquot was assayed 

 for pyruvate — the product of alanine oxidation — with lactic dehy- 

 drogenase and DPNH (.S) . This second assay provided a measure- 

 ment of hydrogen peroxide produced which is independent of the 

 fate of that hydrogen peroxide — either by catalatic or peroxidatic 

 utilization. The entire reaction sequence is outlined in Fig. 3. The 

 second table shows the results of an experiment in which hydrogen 

 peroxide, generated by the enzymatic oxidation of alanine, was trap- 

 ped quantitatively by catalase-ethanol in a reaction mixture capable 

 of oxidative photophosphorylation. The recovery of hydrogen per- 

 oxide by the peroxidative action of catalase is better than eighty 

 per cent complete, as seen in a conqjarison of pyruvate produced with 

 either the oxygen consumed or acetaklehyde produced. This experi- 

 ment shows that the catalase-ethanol trap can give an adequate 

 measure of hydrogen peroxide jjroduction inider the circumstances 

 of the dye-stimulated oxygen exchange. 



