Light scattering application to measurements of the interaction and association of proteins in aqueous solutions with reference to the crystal growth.

测定了不同pH值和不同浓度的胰岛素晶体的形态相图，并对与相图相关的几种溶液进行了静态和动态光散射实验，以考虑光散射技术可以作为快速判断胰岛素结晶是否发生的标准，以及结晶现象与溶液中分子缔合或分子间相互作用的关系。在相图中，我们发现了一些新的现象，即聚集体和液滴的形成，胰岛素晶体在液滴表面的成核和生长，多面体和树枝状胰岛素晶体之间的形态变化。用具有溶解度下液-液相分离曲线的模型相图定性地解释了第一和第二现象。静态光散射实验表明，随着溶液pH值从pH 2增加到pH 7，聚合物的平均分子质量从约6,000增加到约36,000。前者和后者分别相当于胰岛素单体和六聚体。动态光散射法测定的溶液中胰岛素缔合物的平均尺寸也表明，在实验条件下，随着pH的升高，胰岛素缔合作用逐渐增强。结果表明，多面体晶体和树枝状晶体之间的形态变化不能用溶液中胰岛素六角体的量(即晶体生长的有效驱动力)和表观总过饱和度的差异来解释。结论光散射技术有助于识别溶液中胰岛素的缔合，尽管这些技术几乎不能用来判断是否发生结晶。

Determination of the morphological phase diagram of insulin crystal under the various pH and Zn2+ concentration and the static and dynamic light scattering experiments on the several solutions related to the phase diagram have been executed in order to consider that the light scattering technique can be used as a criterion for a quick judgment whether a crystallization of insulin occurs or not and that the relationship between the crystallization phenomena and the molecular association or intermolecular interaction in the solution state.In the diagram, we found several novel phenomena ; i.e., the formation of aggregates and liquid droplets, the nucleation and growth of insulin crystals on the surface of the droplets, and the morphological change between the polyhedral and dendritic insulin crystals. The first and second phenomena were qualitatively explained by using a model phase diagram having a liquid-liquid phase separation curve under solubility. The static light scattering experiments revealed that the average molecular weight increased from ca. 6,000 to ca. 36,000 with increasing the pH of the solution from pH2 to pH7. The former and latter values were equivalent to those of the insulin monomer and hexamer, respectively. The average size of insulin associates in the solution determined by the dynamic light scattering method also indicated that the association of insulin progresses with increasing the pH under the experimental conditions. The results indicated that the morphological change between the polyhedral and dendritic crystals could not be explained by the difference of the amounts of insulin hexamer, i.e. the effective diving force of crystal growth, in the solutions, as well as that of the apparent total super saturation.Conclusively, the light scattering techniques were useful to recognize the association of insulin in the solution, although those were hardly used to judge whether the crystallization occurs or not.