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首页> 外文期刊>Analytical chemistry >Quantitative Analysis and Simultaneous Activity Measurements of Cu, Zn-Superoxide Dismutase in Red Blood Cells by HPLC-ICPMS
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Quantitative Analysis and Simultaneous Activity Measurements of Cu, Zn-Superoxide Dismutase in Red Blood Cells by HPLC-ICPMS

机译：HPLC-ICPMS定量分析和同时测定红细胞中铜，锌超氧化物歧化酶的活性

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摘要

The interest on accurate and precise determination of metalloproteins such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD) involved in the redox balance of living cells is increasing. For this purpose, analytical strategies that provide absolute protein concentration measurements have to be developed. The determination of Cu, Zn-SOD through the measurement of the Cu associated to the protein, which provides its enzymatic activity, by liquid chromatography with online inductively coupled plasma mass spectrometric (ICPMS) detection is described here. Postcolumn isotope dilution analysis (IDA) of Cu has been applied for quantification after evaluation of the column recovery for the total Cu and also Cu-SOD that turned out to be quantitative. When the concentration results obtained via IDA using high-performance liquid chromatography (HPLC)-ICPMS are plotted versus the activity measurements (using the spectrophotometric pyrogallol autoxidation method) a good correlation curve is obtained. Such results permit us, from ICPMS measurements, to obtain simultaneously the Cu, Zn-SOD absolute concentration as well as its enzymatic activity by interpolation in the previously obtained curve. This possibility was explored in real samples (red blood cells of control individuals and patients with metallic total hip arthroplasty) obtaining a good match between direct enzymatic activity measurements and those obtained by interpolation in the correlation curve. The actual protein identification in the red blood cell extract was conducted by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and two matrixes were compared in order to preserve as much as possible the protein-metal interactions during the MALDI process. Interestingly, using a solution containing trihydroxyacetophenone in citrate buffer permitted us to observe some metal-protein interactions in the MS spectrum of the intact Cu, Zn-SOD from red blood cells.
机译：越来越准确地确定与活细胞氧化还原平衡有关的金属蛋白（例如Cu，Zn超氧化物歧化酶（Cu，Zn-SOD））的兴趣。为此，必须开发提供绝对蛋白质浓度测量值的分析策略。本文介绍了通过液相色谱和在线感应耦合等离子体质谱（ICPMS）检测，通过测量与蛋白质相关的铜（提供酶活性）来测定铜，锌-超氧化物歧化酶。在对总铜和证实为定量的Cu-SOD进行柱回收率评估之后，将Cu的柱后同位素稀释分析（IDA）用于定量。将使用高效液相色谱（HPLC）-ICPMS通过IDA获得的浓度结果与活性测量值（使用分光光度的邻苯三酚自氧化法）作图时，可获得良好的相关曲线。这样的结果使我们可以通过ICPMS测量同时通过插值到先前获得的曲线中，同时获得Cu，Zn-SOD的绝对浓度及其酶活性。在真实样本（对照组个体和进行金属全髋关节置换术的患者的红细胞）中探索了这种可能性，从而获得了直接酶活性测量值与通过在相关曲线中内插获得的值之间的良好匹配。红细胞提取物中的实际蛋白质鉴定是通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）进行的，并且比较了两个基质以尽可能保留蛋白质。 MALDI过程中的金属相互作用。有趣的是，在柠檬酸盐缓冲液中使用含有三羟基苯乙酮的溶液，使我们能够观察到红细胞完整的Cu，Zn-SOD的MS光谱中的某些金属-蛋白质相互作用。

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