Quantification of oxidative/nitrosative modification of CYS(34) in human serum albumin using a fluorescence-based SDS-PAGE assay.
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.
Fabisiak
James P
JP
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, 3343 Forbes Avenue, Pittsburgh, PA 15238, USA. fabs@pitt.edu
Sedlov
Andrey
A
Kagan
Valerian E
VE
D001665:Binding Sites; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007700:Kinetics; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D012709:Serum Albumin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D020030:Nitric Oxide Donors; D012709:Serum Albumin; D026422:S-Nitrosoglutathione; D003545:Cysteine