PROTEIN SOLUBILITIES DETERMINED BY A RAPID TECHNIQUE AND MODIFICATION OF THAT TECHNIQUE TO A MICROMETHOD

This laboratory has developed a simple, rapid method for determination of protein solubilities [Pusey and Gernert, J. Crystal Growth 88 (1988) 419]. The system was based upon maximization of the available crystalline surface area and minimization of the free solution volume to be brought into equilibrium. We have determined the tetragonal lysozyme solubility diagram from pH 4.0 to 5.2 (0.1 M sodium acetate), 2%-7% NaCl, 3-25-degrees-C, and portions of the orthorhombic solubility diagram using this technique. Both the tetragonal and orthorhombic solubilities were found to increase smoothly with decreasing salt concentration and increasing temperature. No retrograde solubilities were observed. A major drawback was a requirement for 1-5 mL (ca. 0.5 to 2.5 g) of crystalline protein per column, leading to the devising of micro-methods for implementing this technique. Using column volumes of 75, 300, and 900-mu-L, identical tetragonal lysozyme solubility diagrams (pH 4.5, 0.1M sodium acetate, 5-25-degrees-C) were obtained. Chymotrypsinogen solubilities have also been determined using this apparatus, being retrograde over the temperature range tested. Currently, the primary limiting factor in reducing the crystalline volume is the minimum solution sample size needed to accurately quantitate the protein.