摘要：

The affinities of anti-2,4-dinitrophenyl (DNP) antibodies for a variety of 2,4-dinitrobenzenes have been measured by fluorescence quenching and the values obtained have been verified in representative instances by equilibrium dialysis. All populations of anti-DNP molecules examined, whether isolated from pooled sera or from single bleedings of individual rabbits, were heterogeneous in respect to affinity for dinitrobenzenes. The heterogeneity in virtually every instance could be described by the Sips distribution function over a wide range of binding data, and each titration was thus characterized by an average intrinsic association constant (Ko), and by a heterogeneity index (a). Fractional precipitation of antibodies from serum, by addition of limiting amounts of antigen, yielded from the serum of individual rabbits anti-DNP populations that differed as much as 10,000-fold in Ko. Changes in affinity for ε-DNP-lysine were followed by isolating antibodies from sera at various times after a single injection of DNP-bovine-γ-globulin. The Ko increased progressively with time after immunization and the rate of increase was most conspicuous when small quantities of DNP-bovine-γ-globulin were injected initially. The rise in Ko was markedly delayed when large doses of antigen were injected. Antibody populations isolated early after immunization had nearly the same low affinity for both ε-DNP-L-lysine and for 2,4-dinitroaniline, and it is inferred that their specific binding sites are poorly adapted to the dinitroanilino group and insensitive to the norleucine moiety of ε-DNP-lysine. In contrast, antibodies isolated late after immunization bind ε-DNP-lysine strongly, and from their interactions with a variety of dinitrobenzenes it is inferred that their binding sites, on the average, are just about large enough to accommodate ε-DNP-L-lysine. Complex formation was enthalpy driven: ΔH°values ranged from -8 to -20 kcal mole-1 for different antibody-ligand pairs, and ΔS° values ranged from -5 to -30 entropy units mole-1. When representative 2,4-dinitrobenzenes were bound by anti-DNP molecules, bathychromic and hypochromic spectral shifts were observed; in the case of antibody-bound 2,4-dinitrotoluene a new absorption peak appeared at 300 mμ. The possibility is raised that charge-transfer contributes to the stability of antibody-dinitrophenyl complexes.

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