The binding site of hemoglobin for haptoglobin was investigated through immunological experiments. From the data on Ouchterlony double diffusion reaction of antihemoglobin serum with three test antigens (hemoglobin, hemoglobin-human haptoglobin complex, and hemoglobin-rabbit haptoglobin complex), of anti-(hemoglobin-human haptoglobin complex) serum from which antibodies specific for human haptoglobin were previously excluded by the immunoadsorbent technique with three test antigens, and of anti-(hemoglobin-rabbit haptoglobin complex) serum with three test antigens, it was apparent that antigenic determinants of hemoglobin are not modified nor masked by complex formation with human or rabbit haptoglobin and that new antigenic determinants do not appear in hemoglobin on the complex formation with haptoglobin. Those observations suggest that the binding site of hemoglobin for haptoglobin is quite different from that for antihemoglobin antibody.
The reaction of hemoglobin-human haptoglobin complex with antihemoglobin antibody is much stronger than that of free hemoglobin with same antibody. Hemoglobin-rabbit haptoglobin complex also reacts more strongly with antihemoglobin antibody than free hemoglobin, but the difference is not so marked as for the human moiety. Thus, free hemoglobin, hemoglobin-human haptoglobin complex, and hemoglobin-rabbit haptoglobin complex which all have the same antigenic determinants, showed different reactivity with antihemoglobin antibody. The possible mechanisms for these phenomena are also discussed.
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