Heterogeneous versus bulk nucleation of lysozyme crystals

Abstract

Heterogeneous (on-glass) protein crystal nucleation was separated from the bulk one in systems of thin protein solution layers, confined between two glass plates of custom made quasi two-dimensional all-glass cells, as well as by applying forced protein solution flow. Two commercial samples of hen-egg-white lysozyme, Seikagaku and Sigma were used as model proteins. Applying the classical technique of separation in time of nucleation and growth stages with protein solution layers of thickness 0.05 cm we found that the on-glass crystal nucleation prevailed highly with Seikagaku HEWL, while on the opposite, bulk nucleated crystals represented the main crystal fraction in Sigma solution. Also using 0.05 cm solution layers nucleation rates were measured separately for the on-glass and bulk protein crystals. The process was investigated by varying solution layer thicknesses as well, from 0.05 down to 0.01, 0.0065 and 0.002 cm. Studying the influence of the forced protein solution flow on HEWL crystal nucleation the classical double-pulse technique was modified by separating the nucleation and growth stages not only in time, but simultaneously also in place. In this case we found that the ratio of on-glass formed crystal nuclei to bulk nuclei depended on the flow velocity, but in different manner with Seikagaku HEWL and Sigma HEWL. A plausible explanation of our experimental results is that the bulk crystal nucleation occurs on foreign surfaces as well, e.g. on rests of source biomaterial, which are always present in the protein solutions. Moreover, biomaterial seems to be more active nucleant than glass.

Probabilistic approach to lysozyme crystal nucleation kinetics

“…The activity factor (Table 2, ψ) suggests predominant heterogeneous nucleation, which is in good agreement with the expected natural presence of a variety of impurities in solution samples of commercial lysozyme [19]. The walls of the crystallization solution container have a minor contribution to heterogeneous nucleation, even at a high ratio of wall surface to solution volume [25]. The obtained kinetic parameter ( Table 2, A) is similar to that obtained for lysozyme nucleation at relevant crystallization conditions [13].…”

Controlling protein crystal growth rate by means of temperature

“…[36]: α 0 = 1.35 · 10 −4 K −1 , valid whithin the temperature range: 298 − 323 K. The value obtained for the surface energy: Γ 0 = 5.975gs −2 is compatible with that of the surface tension in a lysozyme solution reported in Ref. [37]: γ ≃ 35gs −2 . For the diffusion coefficient we have obtained D ≃ 1 · 10 −10 µm 2 s −1 .…”

Influence of Mixing Intensity on Lysozyme Crystallization in a Meso Oscillatory Flow Reactor

“…Given the operating conditions and as suggested by the experimental results, it is though that both primary heterogeneous nucleation 40 and attrition-induced secondary nucleation 20,41 may have contributed to the nucleation mechanism. Indeed, the rapid increase in turbidity shown in Figure 2 suggests the formation of a large number of crystals in a short time scale, which has been related in previous works 42−45 with the occurrence of secondary nucleation rather than a sudden increase in primary nucleation.…”