Preparation and properties of cytochrome b 2 from yeast

Abstract

A simple method, which yields about 10 ml. of 1 × 10 −5 M cytochrome b 2 from 100 g. baker's yeast is described. The hematin iron content of the preparation was 0.025 %. The ratio of flavin mononucleotide to heme groups was one to one. The activity was very high. At 3 × 10 −3 M l(+)-lactate, 5 × 10 −5 M ferricytochrome c, and pH 7.4, about 9000 heme groups of cytochrome c were reduced per minute per heme per flavin group of the preparation. The reaction is first order for ferricytochrome c, and the constant is 3.5 × 10 6/sec./mole enzyme. Lowering the pH reduced the reaction speed. A p K of 5.65 with n = 1 is evident. Half-maximal activity was obtained for 1.85 × 10 −5 M ferricytochrome c and excess lactate, or for 3.5 × 10 −4 M l(+)-lactate and excess ferricytochrome c at pH 7.4. The enzyme is specific for l(+)-lactate. d(−)-Lactate does not inhibit the reaction. Versene activates the reaction. Thiol inhibitors and atebrin exert a powerful inactivation of the preparation.