PREPARATION AND CHARACTERIZATION OF LATENT ALPHA(1)-ANTITRYPSIN

Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A be ta-pleated sheet and a mobile reactive center loop. The reactive cente r loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in w hich the reactive center loop is fully inserted into the A sheet of th e molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, ant ithrombin have been shown to adopt the latent conformation. We show he re that the archetypal serpin, alpha(1)-antitrypsin, can also be induc ed to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodiu m chloride concentration on an anion exchange column and has a more ca thodal electrophoretic mobility on non-denaturing polyacrylamide gel e lectrophoresis and isoelectric focusing than native M antitrypsin. Lat ent antitrypsin is inactive as an inhibitor of bovine alpha-chymotryps in, is stable to unfolding with 8 M urea, and is more resistant to hea t-induced loop-sheet polymerization than native but less resistant tha n cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sh eet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from <1% to approximately 35% by refolding from 6 M guanidinium chloride.