Protein Stability and Folding/Unfolding and Formation of Prions and Other Delet

人α -1蛋白酶抑制剂（A1-PI）是一种丝氨酸蛋白酶抑制剂，在体内抑制中性粒细胞弹性酶的活性，在1.4 M Gu时经历胍-盐酸（Gu）诱导的双相展开，处于中间状态。先前，我们证明了在23℃下1.4 M Gu超过1 h，单体和聚合物中间体之间形成了明显的平衡。最近，利用高度纯化的A1-PI， Marszal博士证明了表面上的平衡只涉及单体和二聚体中间体，折叠的二聚体缓慢地聚合形成高分子量聚合物，这似乎是二聚体的多聚体。在折叠单体存在的情况下，该二聚体发生聚合，而单体浓度没有明显变化，从而表明二聚体实际上是聚合单元。一个长期提出的聚合模型假设将A1-PI的柔性反应位点环插入到第二个分子的β片中，以此类推，即单体的环片（或头尾）聚合，形成线性聚合物。为了解释我们的观察结果，我们现在提出二聚体是一种对称的，头对头的二聚体，可能具有互环片性质，通过二聚体表面上相同的疏水斑块（可能是β片结构）的尾对尾接触聚合形成线性聚合物。聚合的环片模型是在热诱导聚合研究的基础上提出的。Marszal博士有初步的、有限的cu诱导和热诱导二聚体的蛋白水解数据，表明这两种二聚体的结构不同，从而表明形成机制也可能不同。自2000年8月开始担任客座助理以来，杜博士开始了一项建立A1-PI CBER参考标准的效价和稳定性的计划，该标准于1988年根据合同生产，但由于效价略低于标签值，因此从未使用过。

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 1.4 M Gu. Previously, we demonstrated that in 1.4 M Gu over 1 h at 23 deg C an apparent equilibrium forms among a monomeric and polymeric intermediates. More recently, with highly purified A1-PI, Dr. Marszal has shown that the apparent equilibrium involves a monomeric and a dimeric intermediate only and that the folded dimer slowly polymerizes to form high molecular weight polymers, which appear to be multimers of dimer. In the presence of folded monomer, this dimer polymerizes with no apparent change in concentration of monomer thereby indicating that dimer is, in fact, is the polymerizing unit. A long proposed model of polymerization hypothesizes the insertion of the flexible, reactive site loop of A1-PI into a beta sheet of a second molecule and so on, i.e. loop-sheet (or head-to-tail) polymerization of monomer, to form linear polymers. To explain our observations, we now propose that the dimer is a symmetrical, head-to-head dimer, possibly of a reciprocal loop-sheet nature, that polymerizes to form linear polymers by tail-to-tail contact presumably of identical hydrophobic patches (possibly beta sheet structures) on the surface of the dimer. The loop-sheet model of polymerization was proposed partly on the basis of studies of thermally induced polymerization. Dr. Marszal has preliminary, limited proteolysis data on Gu-induced and heat-induced dimers that indicate different structures for these two dimers thereby suggesting that the mechanisms of formation may be different also. Since starting as a Visiting Associate in August 2000, Dr. Du has begun a program to establish the potency and stability of the CBER reference standard for A1-PI, which was manufactured under contract in 1988 but which has never been utilized since the potency is somewhat lower than the labeled value.