title: Preparation and characterization of latent alpha 1-antitrypsin.
abstract: Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center 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 which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced 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 sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet 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.
authors.affiliation: Department of Haematology, University of Cambridge, United Kingdom.
authors.forename: D A
authors.initials: DA
authors.lastname: Lomas
authors.forename: P R
authors.initials: PR
authors.lastname: Elliott
authors.forename: W S
authors.initials: WS
authors.lastname: Chang
authors.forename: M R
authors.initials: MR
authors.lastname: Wardell
authors.forename: R W
authors.initials: RW
authors.lastname: Carrell
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012965:Sodium Chloride; D014508:Urea; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D009842:Oligopeptides; D017395:Plasminogen Activator Inhibitor 1; D000515:alpha 1-Antitrypsin; D012965:Sodium Chloride; D014508:Urea; D010047:Ovalbumin