120] DPNH cytochrome c reductase (animal)

Abstract

Publisher Summary This chapter discusses the DPNH cytochrome c reductase for animal. The reduction of ferricytochrome c by DPNH in the presence of the enzyme is measured spectrophotometrically at 550 mμ. The unit of enzyme activity is defined as that amount of enzyme which yields an initial rate of 1.00 per minute at 22°. Specific activity is defined as the number of units per milligram of protein. Protein is determined by the biuret reaction. The assay method is applicable to the determination of reductase activity in homogenates or crude tissue extracts, provided that the following modifications and precautions are observed: (1) the turbidity introduced by the enzyme preparation should not be so great as to make accurate spectrophotometry impossible; (2) the reaction is carried out in 0.05 M phosphate buffer, pH 7.4, in the presence of 0.001 M KCN. The steps described in the purification procedure are: (1) preparation of lyophilized extract, (2) acid ammonium sulfate fractionation, (3) ammonium sulfate fractionation in phosphate at pH 8.0, and (4) refractionation with ammonium sulfate. The purified enzyme is best stored as a solution, 1% in enzyme protein and 1 to 2 % with respect to albumin, in the frozen state at –10°.