ABSTRACT
Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.
Subject(s)
Animals , Humans , Cysteine , Metabolism , Fluorescent Dyes , Nitrosation , Oxidation-Reduction , Proteins , Chemistry , Metabolism , Reactive Nitrogen Species , Metabolism , Reactive Oxygen Species , Metabolism , Sulfenic Acids , Sulfhydryl Compounds , Chemistry , MetabolismABSTRACT
Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80 percent of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25 percent of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.
Subject(s)
Humans , Serum Albumin/chemistry , Serum Albumin/metabolism , Sulfenic Acids/metabolism , Sulfhydryl Compounds/metabolism , Oxidation-Reduction , Protein Conformation , Sulfenic Acids/isolation & purificationABSTRACT
Four spectrophotometric procedures [A-D] for the determination of pantoprazole sodium were investigated. Procedures A and B are stability-indicating methods to determine the intact drug in presence of its degradation products; sulfenamide and sulfenic acid prepared by degrading the pure drug in borate buffer [pH 8] at 37C for 5 days. Procedure A is based on first derivative spectrophotometric measurement in ethanol at 305 nm, whilst procedure B involves coupling of the drug with 2,6-dichloroquinone chlorimide in ethanoldimethylformamide mixture to yield a purple coloured product picked at 515 nm. Procedures C and D depend on charge transfer complexation with dichlorophenol indophenol and dichloronaphthoquinone in CHCl3-methanol and dimethylformamide yielding blue and orange radical anions with lambda max at 640 and 495 nm, respectively. Regression analysis of Beer's plots show good correlations [r=0.9992- 0.9999] and the calibration curves are linear over the ranges of 5-20 mug ml-1, 30-240 mug ml-1, 2-16 mug ml-1 and 40-320 mug ml-1 for procedures A,B,C and D, respectively. The detection limits range between 0.14 and 4.16 mug ml-1. Procedures A and B retain their accuracies in presence of up to 60% and 30% of pantoprazole degradation products, respectively. The average recoveries for commercial tablets are 98.9-100.4%. The results proved to be reproducible and in accordance with those given by a compendial method[11]