Is Agarose an Impurity or an Impurity Filter? In Situ Observation of the Joint Gel/Impurity Effect on Protein Crystal Growth Kinetics.

Abstract

The joint effect of agarose gel and impurities on hen egg white lysozyme crystal growth kinetics was investigated in situ by comparing the two-dimensional (2D) nucleation rate and the step velocity of crystals growing from free and gelled (agarose) solutions having two different levels of purity: highly purified (99.99% pure) and commercial grade (98.5% pure). The 2D nucleation rate and step velocity were measured on {110} faces of tetragonal lysozyme crystals using laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). 2D nucleation rates are enhanced by the presence of gel fibers that act as heterogeneous nucleation sites. These results also show that the specific surface energies are similar for the gel fiber/crystal interface and for the gel fiber/solution interface. This is consistent with the observed incorporation of agarose fibers into the lysozyme crystal lattice and the small effect of gel fibers on step velocity. 2D nucleation in the presence of both gel and impurities is also enhanced but not as much as for gelled purified solutions. The presence of agarose has an almost negligible effect on the step velocity in purified solutions but significantly modifies the step velocity in crystals growing from impure solutions, shifting these values closer to the velocities measured in purified solutions. This velocity increase corresponds to a 7-fold reduction in the concentration of adsorbed impurities at the crystal surface with respect to ungelled experiments. This direct evidence of the diffusive impurity filtering concept is also consistent with the qualitative observations on 2D island morphologies.