Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Protein Chemistry
Methods for Protein Isolation and Purification
Determination of Protein Homogeneity

At The final stage of protein Isolation and Purification, researchers are invariably concerned with the homogeneity of the obtained protein. The homogeneity of an individual protein cannot be evaluated based on a single physicochemical parameter alone; a variety of criteria must be employed. Among the vast array of chromatographic, electrophoretic, chemical, radio- and immunochemical, biological, and sedimentation Methods, the most reliable results for determining protein homogeneity are provided by density gradient ultracentrifugation in sucrose or cesium chloride, Disc Electrophoresis in polyacrylamide gel, isoelectric focusing, immunochemical techniques, and Protein solubility determination. Indeed, if a protein migrates as a single sharp band during gel electrophoresis with its biological activity (enzymatic, hormonal, toxic, etc.) concentrated precisely within this zone, these findings strongly indicate the homogeneity of the studied protein.

The immunochemical method for monitoring the homogeneity of a test protein is based on its precipitation reaction with a specific antiserum obtained from animals immunized with this protein. Strictly proving protein homogeneity requires the simultaneous application of several methods.

The method of protein crystallization using ammonium sulfate, as well as the protein solubility method, has not lost its significance. The latter, originally proposed by J. Northrop*, is based on the Gibbs phase rule, according to which the solubility of a pure substance under given experimental conditions depends solely on Temperature and is independent of The amount of substance present in the solid phase. This method can be performed relatively easily and rapidly on a microscale. Typically, the solubility of increasing amounts of the target protein is determined at a constant volume of solvent.