Use of monoclonal antibodies in the detection of structural alterations occurring in lysozyme on heating.
Seven murine anti-hen egg-white lysozyme (HEL) monoclonal antibodies (MAbs), which recognize distinct epitopes of the native enzyme, were used as macromolecular probes to detect structural or conformational alterations occurring in HEL on heating at 95 degrees C, pH 5. As the interactions of the heat-treated HEL with its corresponding MAbs were carried out at room temperature, only irreversible structural and/or conformational alterations could be detected. The transformation of the native enzyme into its denatured form was followed electrophoretically and chromatographically. The denatured enzyme was more negatively charged at pH 8.4 and exhibited a longer retention time on reverse-phase HPLC than native HEL. Its specific catalytic activity was considerably lower than that of the native enzyme. Of the seven MAbs tested in competitive ELISA assays with native and heat-treated HEL only one, MAb D74.3, failed to recognize the heat-treated enzyme. This antibody, which is directed toward the active site region of the enzyme, was ineffective in inhibiting the catalytic activity of the heat-treated HEL using M. lysodeikticus as substrate. In contrast, the monoclonal antibody D1.3, which recognizes an epitope remote from the active site of HEL, inhibited the catalytic activity of the native as well as the heat-treated enzyme. The results indicate that the active site of HEL undergoes an irreversible structural alteration on heating for 2 hr at 95 degrees C, pH 5. No irreversible structural changes could be detected in the other regions of HEL recognized by the corresponding MAbs.
Kenett
D
D
Department of Biotechnology, Tel-Aviv University, Ramat Aviv, Israel.
Katchalski-Katzir
E
E
Fleminger
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D002645:Chickens; D006358:Hot Temperature; D015394:Molecular Structure; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000276:immunology*
D000911:Antibodies, Monoclonal; D009113:Muramidase