A Procedure for Quantitative Determination of the Diaphorase Activity of Connective Tissue.

D IAPHORASE is a flavin-containing enzyme which catalyzes the oxidation of DPNH (reduced diphosphopyridine nucleotide) in the presence of an electron acceptor. The procedure generally used for determination of the activity of this enzyme is based on spectrophotometric measurement of the reduction of 2,6-dichlorophenolindophenol at 600 mit. As pointed out by Mahler (4), this technic is not applicable to tissue homogenates exhibiting high turbidity. A method was therefore developed for the assay of diaphorase in samples with a high connective-tissue content. Tn that procedure described in the present paper, the decolorization time of 2,6-dichlorophenolindophenol is measured visually in the presence of DPNH and NaCN. The number of Mahier units present in the sample isread from a curve (Fig. 1).The curve was constructed on the basis of decolorization times recorded at 25#{176} with the use of purified commercial diaphorase preparations.* The activities of these preparations were measured spectrophotometrically by Mahier’s (4) method immediately before and after their use in the construction of the