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

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 alpha 1-proteinase inhibitor (alpha 1-PI), alpha 1-antichymotrypsin (alpha 1-ACT), alpha 2-antiplasmin (alpha 2-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 alpha 1-PI and alpha 1-ACT undergo conformational changes following oxidative mixed-disulfide formation and that alpha 2-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 alpha 1-PI was 2-fold faster than that of alpha 1-ACT. However, disulfide formation in alpha 1-PI and alpha 1-ACT was much slower than for any other serpin, e.g., alpha 2-AP and angiotensinogen. We present evidence that alpha 1-PI forms a dimer sensitive to thiol reduction, suggesting cysteinyl-mediated dimerization of alpha 1-PI. The alpha 1-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 cysteinyl232 in alpha 1-PI interacted with the cysteinyl168 of elastase in the proteinase-inhibitor complex. Our data suggests that serpin thiols may play an important role in forming a stable serpin-proteinase complex.