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 35 mg/ml in solutions which contained 0 to 3 M NaCl. Three NaCl concentration ranges, in which the distance between protein molecules varied markedly, were revealed: the distance increased in the range 0 to 0.15 М NaCl, decreased in the range 0.3 М to 1.5 М NaCl and increased again in the range 1.5 М to 3 М NaCl. In lysozyme solutions, which contained 0.3 to 1.5 М 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 3 М 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.

Highlights► Dipole-dipole widening of ESR spectra of spin labeled lysozyme molecule is indicative of protein intermolecular interactions. ► Salt-induced concentration heterogeneity of lysozyme solution has been revealed. ► An analysis of the heterogeneity on the basis of lysozyme phase diagram has been provided. ► A theoretical substantiation of the salt-induced sol-gel transition in protein solution has been described.