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 cry stallizability were investigated by dynamic light scattering and cryst al growth experiments using hen egg-white lysozyme as the model protei n. In the presence of traces of protein impurities, representing no mo re than 2% (w/w) of the total protein, the average diffusion coefficie nts of the macromolecular particles found in undersaturated lysozyme s olutions are significantly lower than those measured with purest lysoz yme 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 autocorrelatio n function. Controlled contamination experiments in which ovalbumin or conalbumin were added to purest lysozyme indicate that aggregates res ult from heterogeneous association of lysozyme molecules with the stru cturally unrelated proteins. These aggregates might become starting po ints for heterogeneous nucleation leading to the growth of ill-shaped microcrystals. Aggregates in under- or supersaturated lysozyme solutio ns containing NaCl can be eliminated by filtration over microporous me mbranes. As a result the number of ill-shaped crystals diminishes dras tically; that of well-shaped tetragonal crystals decreases also but th eir size increases.