Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7559831 | Analytical Biochemistry | 2011 | 8 Pages |
Abstract
The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H2O2) absorbance decrease at 240 nm. 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 5 nM for heCAT, 5 mM for H2O2, and 10 mM for GSH, and the incubation time was 10 min. Nitrite (NO2â) was found to be an uncompetitive inhibitor of heCAT activity (IC50 = 9 μM) and of CAT activity in hemolysate (IC50 â¼Â 750 μM). Nitrate (NO3â) at concentrations up to 100 μM did not inhibit heCAT activity. Azide (N3â) was found to be a very strong inhibitor of the heCAT (IC50 = 0.2 nM) but a relatively weak CAT inhibitor (IC50 â¼Â 10 μM) 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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Anke Böhmer, Jens Jordan, Dimitrios Tsikas,