Formation of New Crystalline Enzymes from Chymotrypsin

A solution of chymotrypsin on slight hydrolysis undergoes an irreversible change into new proteins, two of which are enzymes and have been isolated in crystalline form. The new crystalline enzymes, called beta and gamma chymotrypsins, differ from the original chymotrypsin as well as from each other in many physical and chemical respects, such as molecular weight, crystalline form, solubility, and combining capacity with acid. The new enzymes still possess the same enzymatic properties as chymotrypsin. It thus appears that the irreversible change from chymotrypsin to the new enzymes does not affect the structure responsible for the enzymatic activity of the molecule. The solubility curves of the new enzymes agree approximately with the curves for a solid phase of one component and furnish very good evidence that the preparations represent distinct substances. The various enzymes when mixed at the proper pH have a tendency to form mixed crystals of the solid solution type. Thus at pH 4.0 gamma chymotrypsin combines to form solid solution crystals with either alpha or beta chymotrypsin. Hence at this pH separation of gamma from either alpha or beta by means of fractional crystallization is impossible. At pH 5.0-6.0, however, each material crystallizes in its own characteristic form and at its own rate; thus a fractional separation of the various enzymes from each other becomes feasible.