The utility of photo-affinity labels as `mapping' reagents. A study of sub-populations of a specific rabbit antibody by using structurally related photo-affinity reagents

A specific rabbit antibody against the 4-azido-2-nitrophenyl determinant was photo-labelled by the homologous hapten ε-(4-azido-2-nitrophenyl)-l-lysine, and by the close structural isomer ε-(5-azido-2-nitrophenyl)-l-lysine. The extents of covalent labelling of the antibody-binding site were assessed by using radioactive haptens and exhaustive displacement dialysis, which leaves the unlabelled sites empty but largely intact. A single photolysis of hapten–antibody complex suffices to label those sites that are capable of being labelled. Although there is considerable overlap among sub-populations of antibody that will bind the two haptens non-covalently, sites that can be covalently labelled by one reagent cannot be labelled by the other.