Phase equilibria in the lysozyme-ammonium sulfate-water system

Ternary phase diagrams were measured for lysozyme in ammonium sulfate solut ions at pH values of 4 and 8. Lysozyme, ammonium sulfate, and water mass fr actions were assayed independently by UV spectroscopy, barium chloride titr ation, and lyophilization respectively, with mass balances satisfied to wit hin 1%. Protein crystals, flocs, and gels were obtained in different region s of the phase diagrams, and in some cases growth of crystals from the gel phase or from the supernatant after flee removal was observed. These observ ations, as well as a discontinuity in protein solubility between amorphous flee precipitate and crystal phases, indicate that the crystal phase is the true equilibrium state. The ammonium sulfate was generally found to partit ion unequally between the supernatant and the dense phase, in disagreement with an assumption often made in protein phase equilibrium studies. The res ults demonstrate the potential richness of protein phase diagrams as well a s the uncertainties resulting from slow equilibration. (C) 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 498-506, 2000.