Preparation of a two-disulfide bonded enzymically active derivative from hen egg lysozyme

A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23 degrees for 40 min. The products are reacted with [1-14C]iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-14C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-14C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.