Enzymatic assay of gamma-cystathionase activity using pyruvate oxidase-peroxidase sequential reaction.

摘要

A highly sensitive method has been developed for the determination of gamma-cystathionase (EC. 4.4.1.1.) activity in rat tissues using beta-chloro-L-alanine as a substrate. This method is based on colorimetry for the determination of pyruvate produced from beta-chloro-L-alanine with the beta-elimination catalyzed by gamma-cystathionase, coupling a color enzymatic reaction with pyruvate oxidase and peroxidase. The absorbance increases with the oxidized color of a leuco dye, N-(carboxymethylamino)-4,4'-bis (dimethylamino)-diphenylamine at 727 nm is proportional to the gamma-cystathionase activity. The present method is more sensitive and more rapid than the usual methods and does not require troublesome steps such as centrifugation. The calibration curve is linear up to 1.6 &mgr;g of partially purified enzyme (100 U/l). Comparison with the usual method with L-homoserine as a substrate gave good correlation (r=0.990). The present method was applied to the determination of gamma-cystathionase activity in adult male rat tissues. The mean activities in liver and kidney were 8.03 and 3.91 U/g wet weight (n=10), respectively.