Human alpha-1-proteinase inhibitor (A1-PI), which is a serine protease inhibitor and which in vivo inhibits the activity of neutrophil elastase, undergoes guanidine-HCl (Gu) induced biphasic unfolding with an intermediate state in 1.5 M Gu at 25 deg C. The intermediate state appears transiently stable (over a 1-2 h period) primarily with the formation of monomeric and dimeric intermediates that further polymerize more slowly. Dr. Marszal has folded these smaller intermediates by dilution with buffer and observed that in the mixture of folded species, folded dimer, but not folded monomer, further polymerizes. She has confirmed that this species is dimer by MALDI-TOF measurements and shown that it is disulfide linked (through the single sulfhydryl in each monomer). Dr. Du has established that ca. 80% of the A1-PI monomer has free sulfhydryl and that the dimer exhibits no inhibitory activity. Importantly, Dr. Marszal has demonstrated that the species generated by polymerization of the purified dimer appear to be multiples of it, i.e. no trimer is formed. Furthermore, a reactive center loop (RCL) peptide, which has the same sequence at that of the RCL of A1-PI, which normally targets the active site of neutrophil elastase, binds to the monomer, but not to the dimer, thereby preventing the polymerization of the monomeric intermediate but not of the folded dimer. The inhibition of polymerization of monomer by the peptide has been previously reported and used, in part, to support a model of polymerization involving the insertion of the RCL of one A1-PI molecule between the central strands of a beta-sheet on an adjacent molecule thereby forming a linear chain (i.e. a loop-sheet model for polymerization). Electron micrograph data demonstrate that polymers formed from purified dimer are linear bead-like structures that are similar to those formed from monomer (by heating at elevated temperature) and those isolated from inclusion bodies in hepatocytes from patients with a mutant A1-PI that is poorly secreted. The structural constraints of the disulfide linked dimer and its ability to form linear polymers are difficult to reconcile with the proposed loop-sheet model. We propose that formation of the disulfide linked dimer may involve RCL burial, not insertion, and that polymers of the dimer may form by interaction of beta-sheets exposed on the outer surfaces of the dimer. The model proposed here, involving disulfide linked dimer as the smallest repeating unit (i.e. elementary unit) of the polymer, is an alternate mechanism since under certain reaction conditions, different mechanisms of polymerization may compete. The apparently transiently stable distribution of intermediate species that forms in 1.5 M Gu primarily consists of monomeric and disulfide linked dimeric species. The formation of the disulfide linked dimer species is essentially irreversible and, thus, raises the issue as to why not all of the monomeric intermediate reacts to form dimeric intermediate during the transiently stable period. Dr. Du folded this apparently stable distribution of species and isolated the unreacted A1-PI monomer. By repeating the experiment in 1.5 M Gu with the purified monomer, he demonstrated that the same transiently stable distribution was obtained and thus that microheterogeneity of the A1-PI was not responsible for the failure of all of the monomeric species to form disulfide linked dimer in 1.5 M Gu over 1-2 h. He pursued this investigation by following the time course of polymerization in 1.5 M Gu of cysteine blocked and unblocked A1-PI monomer over period of 4 weeks and found, in fact, for both samples, that eventually almost all the monomeric intermediate disappears with production of dimeric and higher polymeric species. Thus, the apparently transient stable distribution occurs as a result of the kinetics of sequential phases, a faster phase and a much slower phase. Unblocked A1-PI forms non-disulfide linked dimers, which also further polymerize; thus, a comparison of SE-HPLC and non-reducing SDS gel results for the highly polymerized unblocked monomer, which has only 80% free sulfhydryl, will allow an independent estimation of the minimum level of free sulfhydryl. The structure of the non-disulfide linked dimer and its mechanism of polymerization remain to be determined. Beginning is September 2000, Dr. Du began a program to establish the potency and stability of the CBER A1-PI Ref. Standard No. 1. However, after substantial effort in familiarizing himself with the relevant assays, Ref. Standard No. 1 was found to be positive for HIV by NAT although it had been produced commercially in 1988 from plasma that had been screened by a first generation test for HIV. Our goal is to either develop a new reference standard de novo or participate in a WHO collaborative program.
