Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: The albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb‐SS‐X+RSH↔Alb‐SS‐R+XSH) or dethiolation (Alb‐SS‐X+XSH↔Alb‐SH+XSSX), depending on the different pKa values of thiols involved in protein–thiol mixed disulfides (Alb‐SS‐X). It appeared in these reactions that the compound with lower pKa in mixed disulfide was a good leaving group and that the pKa differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb‐TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb‐SS‐X (peaks at 0.25–1 min). In turn, Alb‐SS‐X were dethiolated by the excess nonprotein SH groups because of the lower pKa value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb‐SS‐X was accompanied by formation of XSSX and Alb‐SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma. © 2007 Wiley‐Liss, Inc.