Antigenic sites on cytochrome c 2 from Rhodospirillum rubrum

The antigenic determinants for three monoclonal antibodies against cytochrome c 2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody‐bound cytochrome c 2 and by cross‐reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c 2 . The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the ‘front side’ of the molecule which is not antigenic in mitochondrial cytochrome c 2 . Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome‐ c oxidase bound to the front side of cytochrome c . This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c 2 differs from mitochondrial cytochrome c by an eight‐residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c 2 . Only this antibody cross‐reacted with isolated cytochrome‐ c ‐derived peptides and with apo‐cytochrome c 2 . A preliminary analysis of the polyclonal immune response of five rats against cytochrome c 2 indicates that, unlike in eukaryotic cytochrome c , antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.