Scientia Sinica

74 SCIENTIA SINICA Vol, V

an electron carrier in the assay of the enzyme activity, claimed to have isolated the enzyme in an essentially homogeneous state when examined in the ultracentrifuge. Their enzyme preparation contained no hemin and no significant amount of any of the known water-soluble vitamins except @-lipoic acid™.

We have also been engaged in the isolation and purification of the enzyme and have succeeded in obtaining a preparation which is almost electrophoretically pure and which has a specific activity over twice that reported by Singer, Kearney and Zastrow. This preparation contains iron and a flavin prosthetic group. We are presenting in this paper the detailed method of purification together with some observations on the nature of the prosthetic group and other properties of the enzyme preparation.

Martertacs anp METHOops

Enzymes. Cytochrome c-deficient heart muscle preparation ftom fresh pig hearts was prepared as described by Tsou'*!. Cytochrome ¢ of iron content 0.43% was obtained from pig hearts by the method of Tsou and Li!) D-amino acid oxidase apoenzyme was prepared from pig kidney cortex according to Negelein and Bromel”*! up to step 3 in their paper. Trypsin and chymotrypsin were crystallized from fresh ox pancreas by the method of Kunitz and Northrop"! except that the conversion of trypsinogen to trypsin was carried out according to McDonald and Kunitz'®!, These were the same preparations used five years ago!™!.,

Chemicals. The sources of some of the chemicals used in the present work are as indicated below: potassium ferricyanide, from Schering Kahlbaum, zur Analyse reagent; p-cresol, from Hopkin & Williams, redistilled once before use; 2, 3-dimercaptopropanol, synthesized according to Stocken”! and purified by fractionation under reduced pressure; flavin adenine dinucleotide, prepared from pig heart according to Straub’; calcium phosphate gel, prepared according to Keilin and Hartree’; phenazine methosulphate, synthesized according to Kehrmann and Havas!!; versene (ethylene diamine tetraacetic acid), synthesized according to Smith e¢ al).

Determination of enzyme activity. The decrease in light absorption at 420 mu due to ferricyanide reduction by succinate was used as a measure of the enzyme activity. Unless otherwise specified, the reaction mixture contained: 0.1 M phosphate buffer, pH 7.8, 0.033 M succinate, 5 mM potassium ferricyanide and enzyme solution added at zero time with a total volume of 3.5 ml. After a 10-minute reaction at the desired temperature, 1 ml of 20% trichloroacetic acid was added, and this was followed several minutes later by

*In their recent communication to the 3rd International Congress of Biochemistry, August, 1955 Kearney and Singer have withdrawn their previous statement on the absenc= of water-soluble vitamins in their enzyme preparation, because of the finding of riboflavin in the purified enzyme.