Salt induced thermodynamic instability, concentration heterogeneity and phase transitions in lysozyme solutions

The ESR spin label method was used to estimate an average distance between spin-labeled protein molecules at a concentration of 35mg/ml in solutions which contained 0 to 3M NaCl. Three NaCl concentration ranges, in which the distance between protein molecules varied markedly, were revealed: the distance increased in the range 0 to 0.15М NaCl, decreased in the range 0.3 М to 1.5 М NaCl and increased again in the range 1.5М to 3М NaCl. In lysozyme solutions, which contained 0.3 to 1.5М NaCl, solution heterogeneity was observed to increase gradually during 4 days because of the emergence of supramolecular organization in the form of clusters and aggregates. Viscous gel was formed immediately under salting-out conditions at 3М NaCl, and remained unchanged during 4 days. The results obtained are discussed on the basis of a theoretical and experimental phase diagram of lysozyme solution with an emphasis on analysis of continuous supercritical phase transitions which give rise to various types of dynamic and/or equilibrium protein clusters.