Formation of the four isomers of hen egg white lysozyme containing three native disulfide bonds and one open disulfide bond

Abstract

Reduced partially carboxymethylated hen egg white lysozyme (mucopeptide N-acetylmuramoylhydrolase; EC 3.2.1.17) (approximately 0.8 mol of [1-14C]carboxymethyl groups) was air oxidized at pH 8.0 and 37.degree. C in the presence of 1.5 mM 2-mercaptoethanol for 36 h. Gel filtration of this product gave the lower (native) and higher hydrodynamic volume forms, both containing radioactivity (approximately 35 and 65%, respectively). Ion exchange chromatography of the lower hydrodynamic volume forms yielded renatured lysozyme, 2 major radioactive samples (LHC and LHD) eluting at the positions of monocarboxymethylated lysozyme, and 2 minor radioactive samples eluting at the positions of dicarboxymethylated lysozyme. Sample LHC (approximately 23% of the radioactivity) was essentially homogeneous with respect to electrophoretic mobility, exhibited approximately 39% of the enzymic activity of lysozyme, and contained 0.95 mol of [14C]carboxymethyl groups. Sample LHD (approximately 8% of the radioactivity) was also enzymically active and contained approximately 0.5 mol of [14C]carboxymethyl groups; this low value is apparently due to contamination of noncarboxymethylated species. The radioactive tryptic peptides from samples LHC and LHD were characterized. The results indicated that all 8 isomers, containing 3 presumably native disulfide bonds and 1 free and 1 carboxymethylated sulfhydryl group, are formed on air oxidation of reduced partially carboxymethylated lysozyme. Since in each of these isomers the formation of 1 of the 4 native disulfide bonds is permanently blocked, it would follow that no one of the 4 disulfide bonds of native lysozyme is obligatory in the formation of the other 3 native disulfide bonds.