Determination of γ-Glutamylcysteine Synthetase and Glutathione Synthetase Activity by HPLC

Abstract

The tripeptide glutathione (GSH) in its reduced form is a critical intracellular antioxidant that is essential to many physiological and toxicological processes. The rate-limiting enzyme in intracellular synthesis of GSH is the peptide bond formation between free glutamate and cysteine, mediated via γ-glutamylcysteine (GC) synthetase (EC 6.3.2.2: GCS). GSH synthesis is completed by the addition of glycine to GC, mediated via glutathione synthetase (EC 6.3.2.3; GS). We have developed a rapid and sensitive assay to measure both of these enzymes in the soluble fractions of tissues. For GCS activity, cytosolic fractions of tissue are incubated with an excess of cysteine and glutamate, and the reaction is stopped by addition of sulfosalicylic acid. For GS, dialyzed cytosol is incubated with an excess of GC and glycine. The products of each reaction are derivatized with monobromobimane, and the fluorescent bimane derivatives of glutathione and GC are separated by reverse-phase, ion-pair high performance liquid chromatography and quantified with either ultraviolet (375 nm) or fluorescence (λ_ex = 375 nm, λ_em = 475 nm) detection. Enzymatic formation of products from the respective substrates is linear with time and protein over a wide range. With fluorescence detection, 5 pmol of product can readily be detected.

Key Words: :

• Synthetases
• High performance liquid chromatography
• Glutathione