title: 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 (H(2)O(2)) 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, H(2)O(2)-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, 5mM for H(2)O(2), and 10mM for GSH, and the incubation time was 10 min. Nitrite (NO(2)(-)) was found to be an uncompetitive inhibitor of heCAT activity (IC(50)=9 μM) and of CAT activity in hemolysate (IC(50)∼750 μM). Nitrate (NO(3)(-)) at concentrations up to 100 μM did not inhibit heCAT activity. Azide (N(3)(-)) was found to be a very strong inhibitor of the heCAT (IC(50)=0.2 nM) but a relatively weak CAT inhibitor (IC(50)∼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.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Böhmer
authors.forename: Jens
authors.initials: J
authors.lastname: Jordan
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D001386:Azides / Q000037:antagonists & inhibitors; D002374:Catalase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D005978:Glutathione / Q000032:analysis* / Q000097:blood; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D009573:Nitrites / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D009764:o-Phthalaldehyde / Q000737:chemistry
chemical_list: D001386:Azides; D009573:Nitrites; D009764:o-Phthalaldehyde; D006861:Hydrogen Peroxide; D002374:Catalase; D005978:Glutathione