Hydroxysteroid diaphorase

The same procedure described for -hydroxysteroid dehydrogenase assay can be applied with a few differences [50]. Diaphorase (33 U/ml) is dissolved in 2 ml sodium carbonate-bicarbonate buffer (0.1 M, pH 9.0). Colour reagent is deprived of diaphorase NAD+ is replaced by NADH. The reaction is started by adding 0.1 ml diaphorase. 

Immobilisation of Diaphorase onto Arylamine Glass Beads Dissolved diaphorase (2 ml) is immobilised onto arylamine glass beads (100 mg) through the same method reported for -hydroxysteroid dehydrogenase. The immobilised enzyme is stored in 0.065 M sodium phosphate buffer, pH 7.0, at 4°C. 

Serum or bile samples (0.2 ml) are incubated with 100 mg arylamine glass bound to -hydroxysteroid dehydrogenase, 200 mg arylamine-glass-bound diaphorase and 0.5 ml colour reagent in a 15 ml conical flask, in the dark at 30°C for 15 min (standard assay conditions for mixture of immobilised enzyme) under continuous stirring. The A54o of the reaction mixture is read. 

Rani K, Garg P, Pundir CS (2004) Measurement ofbileacidin serum and bile with arylamine-glass-bound -hydroxysteroid dehydrogenase and diaphorase. Anal Biochem 332 32-37... 

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