The application of four methods for assessing protein homogeneity to crystalline β-lactoglobulin: an anomaly in phase rule solubility tests

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Abstract
A method for fol-lowing attempts at further purifying crystalline beta-lacto-globulin was sought. Ultracentrifugal and immunological (precipitation curve determination) methods tried were unsatisfactory as they showed no evidence of inhomogeneity in the sample tested. Electrophoretic analysis of lactoglobulin was subject to such marked anomalies as to be impractical. "Variable solvent" solubility tests were found most satisfactory, and indicated the presence of 2 main components (ratio approximately 1:4) in lactoglobulin at pH''s 4.5, 4.8 and 5.1. Repeated recrystallizations do not affect this ratio. At pH 5.4 an additional discontinuity occurs in the solubility curves for lactoglobulin associated with a different form of the solid phase (previously amorphous, now crystalline). It is concluded that it does not represent a "true" additional component in the protein. At pH 5.7 the solubility behavior of lactoglobulin is markedly time dependent. The results are shown to be due to progressive crystallization, and provide confirmation for the conclusion that the additional break in the curves at pH 5.4 is due to the taking on by the solid phase of a different (crystalline) form stable, at this pH, only over a limited range of salt concentrations. The ways of detecting this anomalous behavior (when more breaks occur in the solubility curve than there are components in solution) are discussed.
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