Fabisiak James P, Sedlov Andrey, Kagan Valerian E
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, 3343 Forbes Avenue, Pittsburgh, PA 15238, USA.
Antioxid Redox Signal. 2002 Oct;4(5):855-65. doi: 10.1089/152308602760599016.
The SH group represented by cysteine in proteins is fundamental to the redox regulation of protein structure and function. Albumin is the most abundant serum protein whose redox modification modulates its physiologic function, as well as serves as a biomarker of oxidative stress. Measurement of selective Cys modification (S-oxidation/nitrosation, electrophilic substitution) on specific proteins, however, is problematic within complex biological mixtures such as plasma. We have utilized a maleimide fluorogenic SH reagent, ThioGlo-1, to develop a fluorescence-based quantitative assay of SH modification of human serum albumin (hSA) using SDS-PAGE. Fully reduced native albumin containing one free SH (Cys(34)) per molecule was utilized as a model protein to characterize the kinetics of ThioGlo-1 reaction using a solution-based spectrofluorometric assay. Optimum labeling of hSA Cys(34) was achieved within 10 min at 60 degrees C using a threefold molar excess of ThioGlo-1 relative to hSA and required SDS. Comparison of the solution spectrofluorometric assay to fluorescent image analysis of hSA bands localized by SDS-PAGE revealed that SH groups in hSA could be quantified after gel electrophoresis. The solution- and gel-based methods were in excellent concordance in their ability to quantify SH modification of hSA following exposure to phenoxyl radicals and nitric oxide. The application of ThioGlo-1 staining and SDS-PAGE quantified the degree of hSA modification in complex human plasma exposed to oxidative or nitrosative stress and revealed that hSA is more sensitive to S modification than other SH-containing plasma proteins.
蛋白质中由半胱氨酸代表的巯基（SH）基团对于蛋白质结构和功能的氧化还原调节至关重要。白蛋白是血清中含量最丰富的蛋白质，其氧化还原修饰可调节其生理功能，并可作为氧化应激的生物标志物。然而，在诸如血浆等复杂生物混合物中，测量特定蛋白质上的选择性半胱氨酸修饰（S-氧化/亚硝化、亲电取代）存在问题。我们利用一种马来酰亚胺荧光SH试剂ThioGlo-1，开发了一种基于荧光的人血清白蛋白（hSA）SH修饰定量测定方法，采用SDS-PAGE。每个分子含有一个游离SH（半胱氨酸（34））的完全还原天然白蛋白被用作模型蛋白，使用基于溶液的荧光分光光度法表征ThioGlo-1反应的动力学。相对于hSA使用三倍摩尔过量的ThioGlo-1，并需要SDS，在60℃下10分钟内可实现hSA半胱氨酸（34）的最佳标记。将溶液荧光分光光度法与通过SDS-PAGE定位的hSA条带的荧光图像分析进行比较，结果表明，凝胶电泳后可对hSA中的SH基团进行定量。基于溶液和凝胶的方法在定量hSA暴露于苯氧基自由基和一氧化氮后的SH修饰能力方面具有极好的一致性。ThioGlo-1染色和SDS-PAGE的应用定量了暴露于氧化或亚硝化应激的复杂人血浆中hSA的修饰程度，并表明hSA比其他含SH的血浆蛋白对S修饰更敏感。
