[124] Diaphorases

This chapter discusses about the diaphorases. The assay methods involved MB reduction. The DPNH is generated by the interaction of substrate, DPN, and a dehydrogenase and either the Thunberg technique or manometric experiments is used; the disappearance of DPNH spectrophotometrically is followed, using MB as the immediate and O 2 as the final electron acceptor. The method employs measurement of 2,6-di-chlorophenolindophenol reduction at 600 mμ. The method as outlined is directly applicable to use with crude extracts or homogenates provided that (1) the turbidity introduced by the preparation is not so great as to make accurate spectrophotometry impossible and (2) the assay mixture is made 0.001 M with respect to CN – . Under these conditions, the method measures not only diaphorase, if any, but also dye reduction due to DPNH cytochrome reductase. The steps described in the purification procedure are: (1) preparation of homogenate; (2) precipitation at pH 4.6; (3) alcohol extraction; (4) treatment with alumina; (5) heat treatment and first ammonium sulfate fractionation; (6) denaturation by dialysis; and (7) second heat treatment and ammonium sulfate fractionation. The soluble, purified enzyme is specific for DPNH .