Kreilgaard L, Jones L S, Randolph T W, Frokjaer S, Flink J M, Manning M C, Carpenter J F
The Department of Pharmaceutics, Royal Danish School of Pharmacy, Copenhagen.
J Pharm Sci. 1998 Dec;87(12):1597-603. doi: 10.1021/js980126i.
Agitation- and freeze-thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air-liquid and ice-liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against freeze-thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.
搅拌和冻融诱导的重组人因子 XIII（rFXIII）聚集是由于气液和冰液界面处的界面吸附和变性。无论变性刺激如何，聚集途径都是通过可溶性聚集体形成不溶性聚集体。一种非离子表面活性剂，聚氧乙烯山梨醇酐单月桂酸酯（吐温 20），作为表面活性剂浓度的函数，极大地降低了不溶性聚集体的形成速率，从而稳定了天然 rFXIII。最大保护作用出现在接近临界胶束浓度（cmc）的浓度下，与初始蛋白质浓度无关。为了研究表面活性剂诱导稳定化的机制方面，进行了一系列光谱研究。电子顺磁共振光谱表明吐温 20 与 rFXIII 的天然状态或折叠中间状态之间没有发生结合。此外，圆二色光谱表明吐温 20 不能阻止盐酸胍诱导的去折叠所引起的二级结构变化。综上所述，这些结果表明吐温 20 主要通过与应激诱导的可溶性聚集体竞争界面来保护 rFXIII 免受冻融和搅拌诱导的聚集，从而抑制随后向不溶性聚集体的转变。
