Eshdat, Y., Dept. Biophys., Weizmann Inst. Sci., Rehovoth, Israel
McKelvy, J.F., Dept. Biophys., Weizmann Inst. Sci., Rehovoth, Israel
Sharon, N., Dept. Biophys., Weizmann Inst. Sci., Rehovoth, Israel

Hen egg white lysozyme, fully inactivated by the affinity labeling reagent, the 2',3' epoxypropyl β glycoside of di (N acetyl D glucosamine), contained 1 mole of the label per mole of protein. Total enzymatic digestion of the reduced and carboxymethylated affinity labeled enzyme afforded, as a major radioactive product, a compound composed solely of aspartic acid and glucosamine at the molar ratio of 1:2. Peptic digestion afforded a single radioactive peptide corresponding to the sequence Asn(39) Thr Gln Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr(53) in the enzyme. Digestion of this peptide with Clostridium histolyticum aminopeptidase gave a radioactive pentapeptide the composition, partial structure, and other properties of which corresponded to the sequence Gly(49) Ser Thr Asp Tyr(53) in hen egg white lysozyme, and which contained 2 moles of glucosamine per mole of peptide. The authors' findings show that the affinity label is covalently bound to the enzyme via the β carboxyl group of Asp 52 and are in accord with their conclusions from immunochemical and x ray crystallographic studies of the affinity labeled hen egg white lysozyme.