THE ASSAY OF ANIMAL TISSUES FOR RESPIRATORY ENZYMES

Abstract

Until methods for the assay of animal tissues for specific respiratory enzymes have been developed, the brilliant and fundamental researches of Warburg, Meyerhof, Cori, and others cannot be applied to the various aspects of metabolism. By means of the homogenization technique of Potter and Elvehjem (2), it is possible to disperse cellular components so widely that endogenous respiration is virtually stopped due to dilution effects (2), after which particular phases of metabolism may be restored by the addition of the appropriate substrates and soluble cofactors.’ The goal is to isolate a particular reaction and, by so doing, to determine quantitatively the specific enzyme which catalyzes the reaction, rather than to effect a partial isolation of the enzymes by means of fractionations in which the over-all yield is never determined. In the case of the respiratory enzymes, the situation is complicated by the fact that the reaction occurs through the mediation of a series of enzymes, all of which are necessary to obtain oxygen uptake. We have previously presented a method for the quantitative determination of cytochrome c (1) in animal tissues. It was possible in that instance to express the resuhs on an absolute basis; i.e., in gm. or moIes per unit of tissue. In the case of most of the respiratory enzymes, there is no way of expressing the results on an absolute basis, since the measurement is in terms of catalytic effect, and the results must be expressed in the same terms, since no standards are available for comparison. However, by expressing the results in terms of the conventional fJo2, one may compare the results directly with measurements made on tissue slices. This cannot be done when succinic dehydrogenase is measured by the methylene blue technique (3).