An application of RP-HPLC for determination of the activity of cystathionine beta-synthase and gamma-cystathionase in tissue homogenates

摘要

The RP-HPLC-based method of determination of the activity of cystathionine beta-synthase and gamma-cystathionase was undertaken in mouse liver, kidney and brain. Products of the reactions, such as cystathionine, alpha-ketobutyrate, cysteine and glutathione, were measured using the RP-HPLC method. A difference in the cystathionine level between homogenates with totally CTH-inhibiting concentrations of DL-propargylglycine and without the inhibitor was employed to evaluate the activity of cystathionine beta-synthase. Gamma-cystathionase activity was measured using DL-homoserine as a substrate and a sensitive HPLC-based assay to measure alpha-ketobutyrate. The results confirmed high cystathionine beta-synthase activity and no T-cystathionase activity in brain, and high gamma-cystathionase activity in mouse liver. The method presented here allows for evaluating the relative contribution of CBS and CTH to generation of H2S in tissues. Additionally, it provides results, which reflect the redox status (GSH/GSSG) of a tissue. (C) 2014 Elsevier Inc. All rights reserved.