Acetylation of Lysozyme

Abstract

In order to elucidate the significance of amino groups as active groups of lysozyme, egg white lysozyme was acetylated with acetic anhydride. The acetylated lysozyme was fractionated by CM-cellulose column chromatography. All acetylated lysozymes fractionated exhibited 120% relative activity toward glycol chitin at pH 5.6, while the optimum pH shifted to the alkaline side by 0.5 pH units. Trinitrophenylated lysozyme, which was derived from acetylated lysozyme with 1.1 free amino groups per mole and which contained no amino group itself, retained 75% relative activity toward glycol chitin. These results indicate that the amino groups in the lysozyme molecule are not involved in the active site. Acetylated lysozyme behaved the same as the untreated enzyme except the susceptibility to proteolytic digestion. Acetylated lysozyme retained full activity toward glycol chitin. However, lytic activity toward cells of Micrococcus lysodeikticus in neutral media decreased in proportion to the number of amino groups acetylated and its optimum pH shifted to the acid side with increase in the number of acetyl groups introduced. In the acid region below the optimum pH, the lytic activity was the same as that of untreated lysozyme. Very similar results were obtained using cell wall suspension. The first step in lysis of bacterial cells by lysozyme seems to be an interaction between the positive charges of lysozyme and the negative charges on the surface of the bacterial cells. Acetylation of the free amino groups of lysozyme results in a diminution in the numbers of positive charges, causing a decrease in the interaction at neutral pH values. The second step of lysis is hydrolysis of the β-1,4-glucosaminide linkage in the polysaccharide of the cell wall. The last step is dissolution of the damaged cell wall.