PROBING THE CONFORMATION AND ORIENTATION OF ADSORBED PROTEIN USING MONOCLONAL-ANTIBODIES - CYTOCHROME C(3) FILMS ON A MERCURY-ELECTRODE

A novel use of monoclonal antibodies to probe adsorbed protein conform ation is described. Previous electrochemical studies (Zhang, D. B. et al, Anal, Chem, 1994, 66, 3873-3881) described the characteristics of the potential-dependent adsorption of Desulfovibrio vulgaris (Hildenbo rough) cytochrome c(3) on a mercury electrode. Monoclonal antibodies w ere generated with epitopes in the vicinity of heme 1. These antibodie s were utilized to confirm the existence of three conformationally dis tinct electrochemical forms depending upon the applied potentials of o pen circuit (similar to -0.05 V), -0.7 V, and -1.2 V vs AgCl/Ag refere nce. In all three conformations, the cytochrome cg was in a denatured state when compared to soluble protein. When the charge on the electro de was changed from positive to negative (open circuit to -1.2 V), hem e 1 remained oriented toward the solution even though the heme 1 regio n possesses a high positive charge.