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

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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.