Tran S T, Shrake A
Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Arch Biochem Biophys. 2001 Jan 15;385(2):322-31. doi: 10.1006/abbi.2000.2186.
Previous folding studies of alpha-1-proteinase inhibitor (alpha1-PI), which regulates the activity of the serine protease human neutrophil elastase, show an intermediate state at approximately 1.5 M guanidine-HCl (Gu). For the normal form of alpha1-PI, we demonstrate the reversible formation of the same stable distribution of monomeric and polymeric intermediates after approximately 1 h in 1.5 M Gu at approximately 23 degrees C from fully folded or fully unfolded alpha1-PI at similar final total concentrations and show that the stable distribution of monomeric and polymeric intermediates conforms with the law of mass action. We attribute these observations to an apparent equilibrium among intermediates. Our CD data are compatible with the intermediates having slightly relaxed structures relative to that of fully folded alpha1-PI and, thus, with the polymeric intermediates having a loop-sheet structure. Furthermore, we observe that the rates of folding (fast and slow terms) from the intermediate state are the same as those from the fully unfolded state, thereby supporting the contention that this intermediate state is on the folding pathway. We attribute the tendency of the Z mutant protein to polymerize/aggregate to an increased rate of the monomeric intermediate to form the apparent equilibrium distribution of intermediate species relative to its rate of folding to give intact alpha1-PI.
此前对α1-抗胰蛋白酶（α1-PI）的折叠研究表明，它可调节丝氨酸蛋白酶人中性粒细胞弹性蛋白酶的活性，在约1.5 M盐酸胍（Gu）浓度下会出现一种中间状态。对于α1-PI的正常形式，我们证明了在约23℃下，于1.5 M Gu中，从相似最终总浓度的完全折叠或完全展开的α1-PI开始，大约1小时后会可逆地形成相同稳定分布的单体和聚合物中间体，并表明单体和聚合物中间体的稳定分布符合质量作用定律。我们将这些观察结果归因于中间体之间明显的平衡。我们的圆二色性（CD）数据与中间体的结构相对于完全折叠的α1-PI略微松弛相一致，因此也与具有环片层结构的聚合物中间体相一致。此外，我们观察到从中间状态折叠的速率（快和慢项）与从完全展开状态折叠的速率相同，从而支持了这种中间状态处于折叠途径上的观点。我们将Z突变蛋白聚合/聚集的倾向归因于单体中间体形成中间物种表观平衡分布的速率相对于其折叠形成完整α1-PI的速率增加。
