Sulfenic Acid Formation in Human Serum Albumin by Hydrogen Peroxide and Peroxynitrite

Abstract

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises ∼80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M-1 s-1 at pH 7.4 and 37 °C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV−vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with ∼15% decaying after 2 h at 37 °C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in ∼25% of circulating HSA.

Trypanosoma brucei and Trypanosoma cruzi Tryparedoxin Peroxidases Catalytically Detoxify Peroxynitrite via Oxidation of Fast Reacting Thiols

“…5). Under conditions of excess enzyme, the stoichiometry of the reaction was two thiols oxidized by each peroxynitrite added, consistent with the reported stoichiometry for direct thiol oxidation, which involves a two-electron oxidation of the thiol to its sulfenic derivative, an intermediate detected in the case of peroxynitrite-mediated oxidation of the single thiol group of human serum albumin (54). Peroxynitrite-derived radicals CO 3 .…”

Oxidation of the albumin thiol to sulfenic acid and its implications in the intravascular compartment

“…Thus, dimedone (5,5-dimethyl-1,3-cyclohexanedione) selectively derivatizes sulfenic acid yielding a stable thiother, which can be identified by mass spectrometry or by incorporation of a radioactive, fluorescent or biotin tag (41,48). In the case of albumin, the mass spectrometric detection of dimedonetrapped sulfenic acid confirmed that the modification occurred at Cys34 (12).…”

“…The albumin thiol reacts with hydrogen peroxide (H 2 O 2 ) and peroxynitrite (ONOO -) with rate constants of 2.2-2.7 and 3.8 x 10 3 M -1 s -1 , respectively, at pH 7.4 and 37°C (11)(12)(13). These values are similar to free cysteine and glutathione.…”

Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid

“…Titration data were fitted to the Nernst equation using nonlinear regression. The n value was set to 2 as the disulfide-dithiol exchange, and the oxidation and reduction of Cys sulfenic acid are expected to be two-electron transfer processes (35)(36)(37)(38). The redox potential is reported as the mean value Ϯ S.D.…”