PREPARATION OF A TWO-DISULFIDE BONDED ENZYMICALLY ACTIVE DERIVATIVE FROM HEN EGG LYSOZYME

Abstract

A method was developed for preparation of an enzymically active 2-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23.degree. for 40 min. The products are reacted with [1-14C]iodoacetic acid and 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 .apprx. 18% yield. Examination of hydrodynamic volume, tryptophan fluorescence, CD (circular dichroism) and tryptic peptides containing [1-14C] carboxymethyl cysteine indicate that this derivative contains 2 presumably native disulfide bonds and 2 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. The species containing 2 presumably native disulfide bonds and 4 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.