Equilibrium dialysis studies employing -dinitrophenyl-amino caproic acid were conducted with purified grouper antibodies specific for DNP. The 16S antibody preparations obtained at 1 and 2 months of immunization showed heterogeneity of hapten binding indicative of two populations of combining sites. One-half of these sites (an average of four sites per 16S molecule) exhibited an average intrinsic association constant (K_o) of 10⁶M^–1; the K_o of the remaining four-sites was 10⁴M^–1. Thus, the valence of the grouper 16S antibody molecule appears to be eight although the distribution of the high and low K_o sites is unknown, i.e., are they each on the same or on different molecules? The 16S antibody preparations obtained after more prolonged immunization exhibited increasingly lower K_o values; the so-called low K_o sites were no longer detectable. These findings are in contrast to reports of rabbit IgG antibodies showing an increase in K_o with increased time.

The 6.4S antibody preparations obtained from the 1 and 2 month antisera had K_o values of 10⁶M^–1 and a valence of one. These antibodies would not precipitate with antigen. The 6.4S antibody preparations obtained at later times showed decreasing K_o values comparable to those of the 16S antibodies from the same bleedings.

Studies on the thermodynamic parameters of the hapten-antibody interaction showed the grouper 16 and 6.4S antibodies to be similar to each other. These data also showed that the enthalpy and entropy changes of grouper antibody-hapten reactions resemble those reported for rabbit IgG antibodies to this hapten. It is thus suggested that, although considerable evolution of immunoglobulin classes has occurred between fish and rabbits, the antibody combining site may have remained relatively unchanged during a large part of evolutionary time.