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 conformation 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 (Hildenborough) cytochrome c(3) on a mercury electrode. Monoclonal antibodies were generated with epitopes in the vicinity of heme 1. These antibodies were utilized to confirm the existence of three conformationally distinct electrochemical forms depending upon the applied potentials of open circuit (similar to -0.05 V), -0.7 V, and -1.2 V vs AgCl/Ag reference. In all three conformations, the cytochrome cg was in a denatured state when compared to soluble protein. When the charge on the electrode was changed from positive to negative (open circuit to -1.2 V), heme 1 remained oriented toward the solution even though the heme 1 region possesses a high positive charge.