Xia Y, Zweier J L
Department of Medicine, Johns Hopkins Medical Institutions, Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224, USA.
Anal Biochem. 1997 Feb 1;245(1):93-6. doi: 10.1006/abio.1996.9940.
An improved method is reported for measurement of myeloperoxidase (MPO) activity in tissues. While spectrophotometric methods based on oxidation of O-dianisidine or other dyes have been reported for MPO measurement in pure polymorphonuclear leukocytes (PMNs), these methods often fail to accurately assay MPO activity in tissues. We observe that tissue myoglobin or vascular hemoglobin markedly effects the spectrophotometric assay for MPO. Under optimal conditions of 0.53 mM O-dianisidine, 0.15 mM H2O2, pH 6.0, either myoglobin or hemoglobin produced absorbance at 460 nm in a concentration-dependent manner similar to that of MPO. In perfused heart tissue, myoglobin caused a major problem with the assay resulting in an inability to obtain accurate linear results as a function of MPO concentration and PMN number. To eliminate the effect of tissue myoglobin or vascular hemoglobin on the assay, one-step gel filtration chromatography of tissue extracts was introduced. MPO, myoglobin, and hemoglobin were easily separated using a Sephadex G-75 column according to the difference in their molecular weights. A linear relationship between the MPO activity and PMN number was observed only after processing the tissue extracts through the Sephadex G-75 column. Thus, MPO activity in PMN-containing tissues can be precisely quantitated after one-step purification on a molecular exclusion column. Enzyme purification with removal of myoglobin is essential for obtaining accurate measurement of MPO activity and quantitation of PMNs in muscle tissue.
本文报道了一种改进的组织中髓过氧化物酶（MPO）活性的测量方法。虽然已有基于邻联茴香胺或其他染料氧化的分光光度法用于纯多形核白细胞（PMN）中MPO的测量，但这些方法往往无法准确测定组织中的MPO活性。我们观察到组织肌红蛋白或血管血红蛋白会显著影响MPO的分光光度测定。在0.53 mM邻联茴香胺、0.15 mM过氧化氢、pH 6.0的最佳条件下，肌红蛋白或血红蛋白在460 nm处产生的吸光度呈浓度依赖性，与MPO相似。在灌注心脏组织中，肌红蛋白给测定带来了主要问题，导致无法获得作为MPO浓度和PMN数量函数的准确线性结果。为消除组织肌红蛋白或血管血红蛋白对测定的影响，引入了组织提取物的一步凝胶过滤色谱法。根据MPO、肌红蛋白和血红蛋白分子量的差异，使用Sephadex G - 75柱可轻松将它们分离。仅在通过Sephadex G - 75柱处理组织提取物后，才观察到MPO活性与PMN数量之间的线性关系。因此，含PMN组织中的MPO活性在分子排阻柱上一步纯化后可精确定量。去除肌红蛋白进行酶纯化对于准确测量肌肉组织中MPO活性和定量PMN至关重要。
