Liquid–liquid phase separation in supersaturated lysozyme solutions and associated precipitate formation/crystallization

Using cloud point determinations, the phase boundaries (binodals) for metastable liquid–liquid (L–L) separation in supersaturated hen egg white lysozyme solutions with 3%, 5%, and 7% $\mathrm{(w/v)}$ NaCl at $\mathrm{pH}\text{=4.5}$ and protein concentrations $c$ between 40 and 400 mg/ml were determined. The critical temperature for the binodal increased approximately linearly with salt concentration. The coexisting liquid phases both remained supersaturated but differed widely in protein concentration. No salt repartitioning was observed between the initial and the two separated liquid phases. After the L–L separation, due to the presence of the high protein concentration phase, crystallization occurred much more rapidly than in the initial solution. At high initial protein concentrations, a metastable gel phase formed at temperatures above the liquid binodal. Both crystal nucleation and gel formation were accelerated in samples that had been cycled through the binodal. Solutions in the gel and L–L regions yielded various types of precipitates. Based on theoretical considerations, previous observations with other proteins, and our experimental results with lysozyme, a generic phase diagram for globular proteins is put forth. A limited region in the $\mathrm{(T,c)}$ plane favorable for the growth of protein single crystals is delineated.