Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Academic Article


  • 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.
  • Published In

  • Biochemistry  Journal
  • Digital Object Identifier (doi)

    Author List

  • Carballal S; Radi R; Kirk MC; Barnes S; Freeman BA; Alvarez B
  • Start Page

  • 9906
  • End Page

  • 9914
  • Volume

  • 42
  • Issue

  • 33