Institute of Clinical Pharmacology, Hannover Medical School, 30625 Hannover, Germany.
Anal Biochem. 2011 Mar 15;410(2):296-303. doi: 10.1016/j.ab.2010.11.026. Epub 2010 Nov 19.
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.
最常使用的过氧化氢酶 (CAT) 活性测定法基于在 240nm 处测量过氧化氢 (H₂O₂) 吸光度下降的分光光度法。在此，我们报告了一种基于谷胱甘肽 (GSH) 分析的替代高效液相色谱 (HPLC) 测定人红细胞 CAT (heCAT) 活性的方法，该方法作为一种高度稳定、对 H₂O₂ 不敏感的邻苯二醛 (OPA) 衍生物。该方法使用在 pH 7.4 的磷酸盐缓冲盐中的分离的 heCAT 进行开发和验证，并应用于测定裂解的人红细胞中的 CAT 活性。heCAT 活性在初始浓度为 5nM 的 heCAT、5mM 的 H₂O₂和 10mM 的 GSH 下进行测量，孵育时间为 10min。亚硝酸盐 (NO₂(-)) 被发现是 heCAT 活性 (IC₅₀=9μM) 和溶血物中 CAT 活性 (IC₅₀∼750μM) 的非竞争性抑制剂。浓度高达 100μM 的硝酸盐 (NO₃(-)) 不抑制 heCAT 活性。叠氮化物 (N₃(-)) 被发现是 heCAT 的非常强抑制剂 (IC₅₀=0.2nM)，但在人溶血物中是相对较弱的 CAT 抑制剂 (IC₅₀∼10μM)。新型 CAT 活性测定法在更接近细胞中普遍存在的氧化还原条件下进行，并且尽管需要 HPLC 步骤，但仍允许进行高通量分析。
