Potentiometric Determination of Glutathione and Glutathione Transferase Activity

Abstract

A new method of determination of Glutathione Transferase activity (GST) and Glutathione (GSH) in solution has been developed. The determination was performed by detection of ions (H⁺, Cl⁻, F⁻) produced during the GST catalyzed reaction using ion-selective electrodes.

The Michaelis constants of GST π from human placenta toward GSH, 1-Chloro-2, 4-dinitrobenzene (CDNB) and 1-Fluoro-2, 4-dinitrobenzene (FDNB) obtained potentiometrically were similar to those calculated with the classical spectrophotometric method.

The use of pH electrode gave the best results for GST activity (1 pH unit slope per 0.1 unit of enzyme in 1 ml of solution; detection limit of 0.001 unit/ml). Different purification steps of GST TI from human placenta followed by this method gave results comparable to the spectrophotometric one.

The best calibration curve for GSH (2 × M detection limit) was obtained following the production of F- ions with a fixed amount of enzyme at a concentration of 0.2 u/ml. A calibration obtained performing the measurements in filtrated human serum showed the same behaviour.

Key Words:

• Glutathione
• Glutathione Transferase
• Potentiometry
• Ion Selective Electrodes.