Characterization of and mechanism for copper-induced thioureation of serum albumin

Thioureas (Tus) are widely used in chemical and pharmaceutical industries. This study demonstrated that copper induced the disulfide-linkage between Tus, such as α-naphthylthiourea (ANTU) and fluorescein-5-isothiocyanate cadaverine (FTC), with albumin (Alb), a major carrier protein in plasma with multiple functions. This reaction was absolutely copper-dependent, whereas cobalt, nickel, calcium, magnesium, zinc, iron, and manganese ions could not induce the same reaction. The reaction was substrate dose-dependent, and occurred optimally at pH 6.5. The resulting conjugated product was heat-labile, but stable in pH 6.0-8.0 buffer at 25°C. The linkage could be reduced by Cu(I) (in acidic pH) and thiol-reducing agents. The mechanism of albumin thioureation was concluded: (i) the binding of Cu(II) with albumin is not necessary for the reaction, while the formation of Tus-Cu(II) complex is essential; (ii) thioureation resulted from the attack of Tus-Cu(II) at Alb-Cys³⁴-SH to form the Alb-Cys³⁴-S-S-Tus complex accompanied by the release of Cu(I); (iii) the released Cu(I) would back inhibit the reaction because of its competition with Cu(II) for Tus binding. These phenomenons may have important implications for the pharmacokinetics of thiourea-based drugs in plasma.