Dependence of lysozyme growth kinetics on step sources and impurities

Abstract

Interferometric microscopy was used to investigate the growth morphology and kinetics of {110} and {101} faces of tetragonal lysozyme crystals. Solutions were prepared from as-received Sigma and Seikagaku material, and Seikagaku lysozyme further purified by cation exchange liquid chromatography under salt-free conditions. The protein composition of the solutions was characterized by sodium dodecyl sulphate (SDS) electrophoresis with silver staining. We found that on crystals smaller than about 150 μm, 2D nucleation sites were randomly distributed over the faces. With increasing crystal size, surface nucleation became restricted to facet edges and, eventually, to facet corners. This reflects the higher interfacial supersaturation at these locations. However, on some crystals, we observed 2D nucleation at preferred non-corner sites presumably associated with defects. Upon abrupt temperature decreases, dislocation step sources formed on faces that previously had none. Within groups of dislocations, the dominating step source changed frequently. Depending on the activity of the dislocation groups, growth rates of different crystals differed by up to a factor of five during the same experiment. On facets with dislocation step sources, step generation by 2D nucleation became dominant above a critical supersaturation σ∗. In the absence of dislocations, nucleation-induced growth set in at σ < σ∗. In solutions with higher impurity concentrations, the density of the steps generated by 2D nucleation was higher and σ∗ was lower. Hence, it appears that impurity adspecies are active in surface nucleation. The presence of less than 1% of protein impurities with molecular weight (MW) ≥ 30 kD had significant effects on the crystallization kinetics. Step motion was impeded even at high σ, presumably through blocking of kink sites. In solutions without these high MW impurities, facets containing step sources did not grow below σ = ln( C / C sat) < 0.5. In the less pure solutions such a “dead zone” was not observed. Hence, it appears that in lysozyme dead zones are caused by non-protein impurities. In growth from the highly purified material no growth sector boundaries were visible, in contrast to the as-received lysozyme, and striae formation on growth temperature changes appeared drastically reduced.