Enzymic and immunochemical properties of lysozyme—V derivatives modified at lysine residues by guanidination, acetylation, succinylation or maleylation

Abstract

Modification of the amino groups in lysozyme was carried out by guanidination, acetylation, succinylation or maleylation. The specificity of each reaction for amino groups was carefully determined and the enzymic and immunochemical properties of all derivatives studied. Reaction of lysozyme with 1-guanyl-3,5-dimethylpyrazole nitrate gave an electrophoretically homogeneous derivative in which five amino groups were guanidinated. No other amino acids were modified and the derivative exhibited no conformational changes and suffered no loss in enzymic activity, relative to native lysozyme. By acetylation of lysozyme with different amounts of acetic anhydride, two electrophoretically homogeneous derivatives were prepared. Both derivatives were acetylated at all seven amino groups, but one had seven and the other had all fifteen hydroxy amino acids esterified. Reaction of lysozyme with succinic or maleic anhydride resulted in electrophoretically heterogeneous preparations even when all amino groups were modified. There was also a non-uniform esterification of aliphatic hydroxy amino acids. The acetylated, succinylated, or maleylated derivatives showed small, but measurable conformational changes. The enzymic activity was completely eliminated upon acylation of four or more amino groups and this loss was attributed to changes in electrostatic interactions between the modified enzyme and the negatively charged bacterial cell wall. With antisera to lysozyme, the guanidinated derivative possessed full (100 per cent) antigenic reactivity, relative to lysozyme. The two acetylated preparations retained substantial, and comparable, antigenic reactivity with these antisera. In succinylated or maleylated derivatives, the electrophoretic heterogeneity was not accompanied by immunochemical heterogeneity. A considerable amount of cross-reactivity was retained, even when all amino groups were succinylated or maleylated. Partially or completely succinylated lysozymes were antigenic in rabbits and antibodies to the completely succinylated derivative exhibited (a) a specificity against lysozyme, (b) a specificity against antigenic regions containing succinyl groups and showing carrier specificity and (c) a haptenic specificity against succinyl groups attached to unrelated carrier proteins. From these findings it was concluded that retention of the charge of certain amino groups is essential for intact antigenic reactivity. Some amino groups (possibly four) and at least eight aliphatic hydroxyl groups are not located within antigenic sites of lysozyme. Furthermore, succinyl and maleyl groups are immunochemically distinct.