Enzymic and immunochemical properties of lysozyme—VIII. Modification of two carboxyl groyps and their role in the antigenic reactivity

Abstract

Reaction of lysozyme with carbodiimide followed by coupling with glycine methyl ester (GME) or with histidine methyl ester (HME) resulted in the preparation of two derivatives. After column chromatgoraphy the two derivates were homogeneous by starch gel electrophoresis. Both derivatives were modified at aspartic acid 119 and at the carboxyl group of the end-chain leucine residue. However, the one derivative the two carboxyl groups were coupled to GME (GME 2-Lysoz) and in the other, these two carboxyl groups were couple to HME (i.e. HME 2-Lysoz). Each derivative suffered a dreastic loss in enzymic activity, most likely as a result of conformational changes which accompained the modification. HNE 2-Lysoz showed a greater loss in lytic activity than GME 2-Lysoz. With antisera to lysozyme, GME 2-Lysoz possessed equal antigenci reactivity ot that of the homologous antigen, while HME 2-Lysoz suffered an appreciable decrease in reactivity. Antisera were also raised against GME 2-Lysoz and with these antisera, lysozyme and GME 2-lysoz possessed equal antigenic reactivities. On the other hand, HME 2-Lysoz showed lower antigenic reactivity. From these results and previously reported conformational studies, which showed very slight conformational changes in GME 2-Lysoz and large changes in HME 2-Lysoz, it was concluded that aspartic acid 119 and the C-terminal leucine residue are not essential parts of an antigenic reactive region in lysozyme.