High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative

Abstract

The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H2O2) absorbance decrease at 240nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H2O2-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5nM for heCAT, 5mM for H2O2, and 10mM for GSH, and the incubation time was 10min. Nitrite (NO2−) was found to be an uncompetitive inhibitor of heCAT activity (IC50=9μM) and of CAT activity in hemolysate (IC50∼750μM). Nitrate (NO3−) at concentrations up to 100μM did not inhibit heCAT activity. Azide (N3−) was found to be a very strong inhibitor of the heCAT (IC50=0.2nM) but a relatively weak CAT inhibitor (IC50∼10μM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.