Role of oxidative mixed-disulfide formation in elastase–serine proteinase inhibitor (serpin) complex

Abstract

To understand the role of thiol and oxidative mixed-disulfide exchange reaction in serpins, we analyzed the conformation of native and mixed-disulfide forms of α1-proteinase inhibitor (α1PI), α1-antichymotrypsin (α1-ACT), α2-antiplasmin (α2-AP), angiotensinogen, and ovalbumin. The conformation of native and oxidized mixed-disulfide serpins was measured by transverse urea gradient (TUG) gels. The results suggest that the acute phase proteins α1-PI and α1-ACT undergo conformational changes following oxidative mixed-disulfide formation and that α2-AP and angiotensinogen do not. The kinetics of disulfide formation was followed by measuring changes in absorbance at 412 nm resulting from Ellman's reaction of disulfide exchange. The rate of mixed-disulfide formation in albumin was 10-fold faster than in the serpin tested. The rate of disulfide exchange in α1-PI was 2-fold faster than that of α1-ACT. However, disulfide formation in α1-PI and α1-ACT was much slower than for any other serpin, e.g., α2-AP and angiotensinogen. We present evidence that α1-PI forms a dimer sensitive to thiol reduction, suggesting cysteinyl-mediated dimerization of α1-PI. The α1-PI also demonstrated two types of inter-protein disulfide linkages: one resulting in homodimer and other involving heterodimer formation. TUG–Western immunoblot methodology was developed to identify the conformational changes in serpins. We found that the conformational changes in serpins by mixed-disulfide formation are due to unfolding and not to decomposition or degradation in TUG gels. Using fluorescence measurements with isolated tryptic fragments of fluorescence-labelled elastase, we observed that the cysteinyl232in α1-PI interacted with the cysteinyl168of elastase in the proteinase–inhibitor complex. Our data suggests that serpin thiols may play an important role in forming a stable serpin–proteinase complex.Key words: serpin, extracellular matrix, proteinase, reduction, oxidation, inhibitor.