The Preparation, Properties, and Reactivation of the Mixed Disulfide Derivative of Egg White Lysozyme and l-Cystine

Abstract

Abstract The mixed disulfide of lysozyme and cystine can be prepared by reaction of fully reduced lysozyme and cystine at pH 9.5. The mixed disulfide is enzymatically inactive and contains 16 residues of half-cystine per molecule. On reaction with cysteine, mercaptoethylamine, or mercaptoethanol, the mixed disulfide can be converted to active lysozyme. The initial rate of reactivation varies with pH, temperature, concentration of disulfide, and concentration of mercaptan; under the best conditions, 50% of the original activity is regenerated in 6 to 7 min. The extent of reactivation is generally between 70 and 85% under a variety of conditions. On the basis of kinetic measurements and characterization of the lysozyme formed throughout the reactivation process, a tentative mechanism for re-formation of the correct disulfide bonds in lysozyme has been proposed. Chromatographic analysis of the reactivated enzyme shows that it contains two major components. One chromatographic component is indistinguishable from native lysozyme, but the other has about 50 to 60% of the specific activity of native lysozyme and it is not converted to native lysozyme on further reduction and oxidation.