Catalytic antibodies. Circular dichroism and UV-vis studies of antibody-metalloporphyrin interactions

Abstract

Circular dichroism and UV-vis measurements were used to study the interaction between several water-soluble metalloporphyrins and monoclonal antibodies (MABs) that were elicited against a structurally related dihydroxytin(IV) porphyrin, la. Some of the MAB-metalloporphyrin complexes studied were previously shown to mimic hemoprotein-like activity, such as catalytic epoxidation of styrene by iodosobenzene. MAB-metalloporphyrin complex formation is usually accompanied by significant bathochromic shift and hypo-/hyperchromicity changes of the absorption maxima in the porphyrin Soret band region. Induced CD spectra in the same region (350-450 nm) are detected upon complex formation. They follow Beer’s law, exhibit isosbestic behavior, and show no significant change in the general shape of the Cotton effect as a function of the antibody:porphyrin ratio. Signal intensity (ellipticity) increases with increasing antibody:metalloporphyrin ratio, reaching an upper limit that represents saturation complexation, all indicating a specific 1:l binding phenomenon. Strong dependence of both signal shape and signal intensity upon the specific metal and antibody were observed, suggesting that induced CD may serve as a powerful tool for the unequivocal differentiation of two seemingly identical MAB clones. In cases of intense induced CD signals, it is possible to obtain MAB-metalloporphyrin dissociation constants using a titration curve where ellipticity is measured as a function of the antibody:porphyrin ratio and presented as a Scatchard plot. Both the UV-vis absorbance and induced CD spectrum of the metalloporphyrin-MAB complex remain unchanged over a broad pH range between 6 and 11, indicating remarkable stability of these complexes and reflecting the dominant role of electrostatic interactions between the hapten carboxylate groups and the antibody combining site.