Studies on the Combining Site of Anti-p-Azobenzoate Antibody

Abstract

Summary The effect of acetylation on the combining region of specifically purified anti-p-azobenzoate antibody was investigated by measuring the binding of hapten by antibody—a method which provides direct information as to the number of combining sites remaining. The use of small amounts of reagent resulted in loss of precipitating activity without a parallel decrease in binding capacity, indicating that most or all of the resulting nonprecipitating antibodies were still bivalent. The sma results were obtained for the binding of radioactive p-iodobenzoate or p-(p-hydroxyphenylaxo)-benzoate. The failure to precipitate is attributed to electrostaci repulsion arising from the increased negative charge of the acetylated antibody. The use of considerably larger amounts of acetic anhydride brought about a loss of binding, as well as precipitating acitvity. This loss was prevented by the presence of homologous hapten, demonstrating that the reagent attacks the combining region of the antibody. The loss of precipitating activity, however, awas not prevented by hapten, in accord with the posulate that the loss of precipitating and binding capacities occur through different mechanisms. Even in the absence of hapten, over 90% of the amino groups of the molecule could be acetylated without much loss of binding capacity. This suggests either that a group in th combining region toher than an amino group is attacked by acetic anhydride at neutral pH; or if an amino groups is involved it is exceptionally unreactive.