Determination of protein crystallization kinetics by a through-flow small-angle X-ray scattering method

Abstract

The kinetics of protein crystallization was analyzed using a modified Small-Angle X-ray Scattering (SAXS) setup that was operated in a flow-through mode. The model protein for the study was lysozyme, which was crystallized from supersaturated aqueous solutions of ammonium sulfate. A small amount of the protein solution was forced to recirculate within the SAXS setup. The SAXS intensity curves were measured at different time intervals and converted into kinetic profiles using a calibration factor. The diffraction images of the sample taken at the end of the kinetic experiments were recorded to confirm the presence of the crystalline phase. A population balance model was used to describe the course of the crystallization process. The results of the measurements and numerical simulations were compared to those obtained from a typical stirred-tank crystallizer (STC), where seeded and unseeded crystallization was performed. The kinetic data acquired from the SAXS and STC setups followed similar trends regarding kinetic dependencies on the salt concentration and the supersaturation degree. Moreover, the hydrodynamic conditions in the SAXS setup were determined, for which a quantitative agreement between the kinetic data acquired from both systems was achieved. Then, the same kinetic equations could be used to describe the course of crystallization in both systems. Hence, the modified SAXS technique can potentially substitute the STC-based method in quantification of crystallization kinetics.