Assignment of a single disulphide bridge in human α2-antiplasmin: implications for the structural and functional properties

Human α₂-antiplasmin (α₂AP) is a serpin involved in the regulation of blood coagulation. Most serpins, unlike smaller serine proteinase inhibitors, do not contain disulphide bridges. α₂AP is an exception from this generalization and has previously been shown to contain four Cys residues organized into two disulphide bridges [Lijnen, Holmes, van Hoef, Wiman, Rodriguez and Collen (1987) Eur. J. Biochem. 166, 565–574]. However, we found that α₂AP incorporates iodo[¹⁴C]acetic acid, suggesting that the protein contains reactive thiol groups. This observation prompted a re-examination of the state of the thiol groups, which revealed (i) a disulphide bridge between Cys⁴³ and Cys¹¹⁶, (ii) that Cys⁷⁶ is bound to a cysteinyl-glycine dipeptide, and (iii) and Cys¹²⁵ exists as either a free thiol or in a mixed disulphide with another Cys residue. The disulphide identified between Cys⁴³ and Cys¹¹⁶ appears to be conserved in orthologous proteins since the homologous Cys residues form disulphide bonds in bovine and possibly mouse α₂AP. The conservation of this disulphide bridge suggests that it is important for functional aspects of α₂AP. However, the structural and functional analysis described in this study does not support this conclusion.