The Reactions of Antibodies with Hemeprotein Antigens

When an antibody combines with a hemeprotein antigen the fluorescence of the tryptophan residues of the antibody is partially quenched. With fluorescence quenching as an indicator, the stoichiometries and kinetics of the reactions of antihemoglobin A antibodies with various hemoglobins and the reactions of antihuman cytochrome c antibodies with various cytochromes c have been studied. The differences in antigenic specificity between hemoglobins A1, Lepore, and A2 and between human and monkey cytochrome c are easily detected. The data indicate that the number of antigenic sites on a protein is limited and is the factor which controls the number of antibodies bound per antigen molecule. These antigenic sites differ in the rate at which they combine with their respective antibodies, but the second order rate constants for these reactions are about 106 m-1 sec-1.