PROTEIN STABILITY AND FOLDING/UNFOLDING AND FORMATION OF PRIONS AND OTHER DELETER

人α -1蛋白酶抑制剂（a-1- 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1.5 M Gu时经历胍-盐酸（Gu）诱导的双相展开，并处于中间状态。之前，我们证明了这种中间态可以由一个或多个折叠中间体组成。1.5 M - Gu的尺寸不排除高效液相色谱显示，在25℃下，单体和聚合物中间体在2小时内形成平衡分布。这些聚合物中间体主要是二聚体、三聚体和四聚体，与一些更高的聚合物具有环片性质（即通常靶向弹性蛋白酶活性位点的反应位点环插入在另一个a -1- pi分子的β片区域的中心链之间）。从1.5 M Gu稀释折叠得到反映中间体的活性单体和非活性聚合物的水平。动力学研究表明，这种平衡状态在折叠途径上，从完全展开的状态折叠是在动力学控制下的，从而允许形成不稳定但活性单体。从今年1月开始，Marszal博士发现，在1.5 M Gu中观察到的相对较快的聚合之后，在25摄氏度的一段时间内，会发生一种慢得多、有序度更高的聚合。在1.5 M Gu中的中间体和由1.5 M Gu折叠形成的单体和聚合物中都观察到这种较慢的聚集，尽管后者较慢。在串联TSK 3000柱的空隙体积中形成洗脱液的聚合物。Marszal博士已经证明，这种高阶聚集体可以解离成具有高Gu的单体。她正在描述这种高阶聚合体的性质，这种聚合体可能不是环状的。一个有趣的可能性是，1.5 M Gu中的快速聚合可能与突变A-1-PI患者的分泌缺陷有关，而这种突变A-1-PI在合成部位肝细胞中形成包涵体的高阶聚集速度较慢。在确定a -1- pi样品的特定抑制活性（效力）时，Marszal博士利用胰蛋白酶作为目标蛋白酶，为此她使用显色活性位点滴定剂准确测量了活性酶的浓度。她观察到，在低胰蛋白酶和低A-1-PI浓度下，A-1-PI的结合不是化学计量的，因此表明抑制常数低于假设。因此，她计划确定该常数，以建立测定活性A-1-PI浓度的最佳条件，从而能够准确地重新测定CBER A-1-PI参考标准品的活性浓度，目前估计该标准品的效价约为其最初标记效价的75%。本标准的稳定性将在效价方面进行监测。

Human alpha-1-proteinase inhibitor (A-1-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. Previously, we demonstrated that this intermediate state can consist of one or more folding intermediates. Size exclusion-HPLC in 1.5 M Gu shows that an equilibrium distribution of monomeric and polymeric intermediates forms over 2 h at 25 deg C. These polymeric intermediates are primarily dimeric, trimeric, and tetrameric with some higher polymers and are of a loop-sheet nature (i.e the reactive site loop, which normally targets the active site of elastase, is inserted between the central strands of a beta sheet region on another A-1-PI molecule). Folding by dilution from 1.5 M Gu gives levels of active monomer and inactive polymer that reflect those of the intermediates. Kinetic studies demonstrate that this equilibrium state is on the folding pathway and that folding from the fully unfolded state is under kinetic control thereby allowing the formation of the less stable but active monomer. Since starting in January, Dr. Marszal has discovered a much slower, higher order aggregation that occurs at 25 deg C over a period of days after the relatively rapid polymerization observed in 1.5 M Gu. This slower aggregation is observed both for the intermediates in 1.5 M Gu and for monomer and polymers formed from folding from 1.5 M Gu although the latter is slower. The polymer that forms elutes in the void volume of tandem TSK 3000 columns. Dr. Marszal has demonstrated that this high-order aggregate may be dissociated to monomer with high Gu. She is in the process of characterizing the nature of this high-order aggregate, which may not be of a loop-sheet nature. An interesting possibility is that the rapid polymerization in 1.5 M Gu may relate to the secretion defect in patients with mutant A-1-PI and the slower high-order aggregation to the formation of inclusion bodies of this mutant A-1-PI in the hepatocytes, the site of synthesis. In determining the specific inhibitory activity (potency) of samples of A-1-PI, Dr. Marszal utilized trypsin as the target protease, for which she accurately measured the concentration of the active enzyme with a chromogenic active site titrant. She has observed that at low trypsin and low A-1-PI concentrations, the binding of A-1-PI is not stoichiometric thereby indicating that the inhibition constant is lower than assumed. Thus, she plans to determine this constant in order to establish optimal conditions for determining the concentration of active A-1-PI thereby allowing an accurate redetermination of the active concentration of the CBER A-1-PI Reference Standard, which currently is estimated to have approximately 75% of its originally labeled potency. The stability of this standard will be monitored in terms of potency.