Assay of the glutathione-synthesizing enzymes by high-performance liquid chromatography

We report a new convenient assay of the activity of gamma-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in crude microbial extracts as well as in purified enzyme preparations. The assay is based on the quantitative analysis of the reaction products by high-performance liquid chromatography after derivatization of the thiol group with 5,5'-dithiobis-(2-nitrobenzoic acid) as described by J. Reeve, J. Kuhlenkamp, and N. Kaplowitz [(1980) J. Chromatogr. 194, 424-428]. In addition, the procedure yields information on basal levels of gamma-glutamylcysteine and glutathione in crude microbial extracts. The two enzymes responsible for glutathione biosynthesis can be determined in parallel under the same chromatographic conditions. No prior separation from substrates and by-products is necessary. Product formation is linear with time for at least 30 min between 0.03 and 12 mU for both enzymes. Even in crude extracts 0.2-0.5 nmol of products formed can be detected with certainty. The method was found to be sensitive and highly reproducible.