Interaction of human hemoglobin with haptoglobin or antihemoglobin antibody

The physicochemical and biochemical properties of hemoglobin associated with haptoglobin were compared with those of hemoglobin bound by antihemoglobin antibody. The mechanism of enhanced peroxidase activity of hemoglobin bound by haptoglobin was concluded not to be activation but stabilization of hemoglobin at acidic pH by haptoglobin. Haptoglobin protects hemoglobin from denaturation by acid, and moreover it can regenerate denatured hemoglobin at acidic pH. Specific antibody, on the other hand, does not enhance the peroxidase activity of hemoglobin, nor does it prevent the acid denaturation of hemoglobin. Hydrogen peroxide peroxidase complex (Compound I), which has not yet been detected in the H₂O₂ hemoglobin system, was observed in the H₂O₂ hemoglobin haptoglobin complex system, indicating that haptoglobin stabilized the H₂O₂ hemoglobin complex. Hemoglobin bound by antibody shows a higher affinity for oxygen than free hemoglobin (p₅₀ = 2.0 mm Hg at pH 7.0, and p₅₀ = 3.0 mm Hg at pH 7.4), a biphasic Hill plot, and slight preservation of heme heme interaction (n = 1.6 at y/1 - y > 1.5, and n = 1.0 at y/1 -y < 1.5) and somewhat reduced Bohr effect (r = -0.37). These characteristic functions of hemoglobin bound by antibody are in striking contrast to those of hemoglobin bound by haptoglobin. Hemes of hemoglobin antibody complex are not degraded by dithionite under aerobic conditions, whereas those of hemoglobin haptoglobin complex are degraded, being consistent with the view that hemoglobin bound by antibody is tetrameric, whereas hemoglibin bound by haptoglobin is dissociated. The binding site of hemoglobin for haptoglobin was investigated through immunological experiments. From the data on the immune precipitation reaction of antihemoglobin serum in gels with free hemoglobin, hemoglobin haptoglobin (human) complex, and hemoglobin haptoglobin (rabbit) complex, it was apparent that the antigenic determinants of hemoglobin were not modified nor masked, and moreover new antigenic determinants of hemoglobin did not appear on complex formation with haptoglobin. These observations suggest that the binding site of hemoglobin for haptoglobin is quite different from that for antihemoglobin antibody.