EFFECT OF MACROMOLECULAR IMPURITIES ON LYSOZYME SOLUBILITY AND CRYSTALLIZABILITY - DYNAMIC LIGHT-SCATTERING, PHASE-DIAGRAM, AND CRYSTAL-GROWTH STUDIES

The effects of macromolecular impurities on protein solubility and crystallizability were investigated by dynamic light scattering and crystal growth experiments using hen egg-white lysozyme as the model protein. In the presence of traces of protein impurities, representing no more than 2% (w/w) of the total protein, the average diffusion coefficients of the macromolecular particles found in undersaturated lysozyme solutions are significantly lower than those measured with purest lysozyme preparations. This fact is explained by the simultaneous existence of individual molecules and of large size aggregates in contaminated solutions, as indicated by the bimodal light scattering autocorrelation function. Controlled contamination experiments in which ovalbumin or conalbumin were added to purest lysozyme indicate that aggregates result from heterogeneous association of lysozyme molecules with the structurally unrelated proteins. These aggregates might become starting points for heterogeneous nucleation leading to the growth of ill-shaped microcrystals. Aggregates in under- or supersaturated lysozyme solutions containing NaCl can be eliminated by filtration over microporous membranes. As a result the number of ill-shaped crystals diminishes drastically; that of well-shaped tetragonal crystals decreases also but their size increases.