Reactivity of the Thiol Group in Human and Bovine Albumin at pH 3–9, as Measured by Exchange with 2,2′‐Dithiodipyridine

Abstract

The kinetics for exchange between an aromatic disulphide and the thiol group in human and bovine albumin as well as in glutathione were investigated in the pH range 2.5–9.8. For both albumins the rate constants exhibit a maximum near pH 3, confirming the results of Svenson and Carlsson's investigation of bovine albumin [A. Svenson and J. Carlsson (1975) Biochim. Biophys Acta, 400, 433–438]. This was related to the well known N–F conformational change of the protein. At pH 5–8 the reactivity of the thiol group in both albumins and glutathione changes sharply, probably due to ionization of the thiol group. At pH above 8, however, the reactivity of the thiol group in albumins, but not in glutathione, becomes nearly independent of pH. In addition, a conformational change at pH 6.5–8.5 was studied by means of differential spectroscopy of bilirubin, liganded to human albumin. This neutral transition appeared to proceed identically in mercaptalbumin and nonmercaptalbumin. It is concluded that (a) the pK of the thiol group in albumin is significantly below that of SH in glutathione, and (b) ionization of this thiol group, Cys‐34, is independent of the neutral transition.

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

“…To speed up the oxidation reaction as well as to allow direct monitoring of the reaction, the thiol groups were activated with 2,2′-dithiodipyridine (DTDP) (Grassetti and Murray 1967;Pedersen and Jacobsen 1980;Riener et al 2002) (Fig. 1a).…”

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

“…In addition, the two close groups of His39 and Tyr84 affect the reactivity of the thiol (16). Also, HSA presents pH-dependent structural transitions that impact on thiol reactivity (17). Not surprisingly then, the acidity constant value (pK A ) of the albumin thiol remains controversial.…”

The thiol of human serum albumin: Acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid

“…Values ranging from 5 to 8.8 have been reported [32][33][34][35], sometimes as the result of assuming equivalency between the properties of the free thiol of bovine and human albumins, an association recently shown not to apply in solution [36,37]. Previous experiments performed by some of us analyzing the pH dependence of the reactivity towards H2O2, peroxynitrite, or DTNB have yielded values in the range 7.8-8.6 [24,26].…”

“…Such increase can be ascribed to a conformational change of the protein known as the N-F transition that originates in the protonation of residues bearing carboxylate groups. Furthermore, HSA can undergo another conformational transition at pH 8-9, the N-B transition [32]. Fourth, not only the Cys34 thiol does ionize but also other neighboring residues do (e.g.…”

“…Second, according to 1 H-NMR experiments, the Cys34 sulfur can alternate between buried and exposed conformations, shifting among them depending on its redox and chemical modification states [30,31]. Third, HSA suffers pH-dependent conformational transitions that can potentially impact on its thiol reactivity [1,32,33]. For example, for its reaction with 2,2-dithiodipyridine a rate increase attributable to thiol deprotonation is found at pH > 7.…”