Interactions of Porphyrins with Rabbit Hemopexin

The formation of complexes of rabbit hemopexin with a representative group of porphyrins was investigated to determine the stoichiometry and estimated dissociation constants (Kd) as well as the effect of pH on these interactions. In a 1:1 molar ratio, rabbit apohemopexin (RHx) binds iron-protoporphyrin IX (heme), iron-deuteroporphyrin IX (deuteroheme), iron-mesoporphyrin IX (mesoheme), and cobalt-deuteroporphyrin IX. The estimated Kd values are heme ≃ mesoheme ≃ deuteroheme ≤10-8m; and cobalt-deuteroporphyrin ≤10-7m. The relative affinity of RHx was shown to be deuteroheme > nickel-deuteroporphyrin IX > deuteroporphyrin IX, and cobalt-deuteroporphyrin > nickel-deuteroporphyrin, by displacement of ligands with lower affinity employing difference spectrophotometry or gel electrophoresis. The binding strength was influenced more by the presence and nature of the metal chelated to the porphyrin nitrogens than by the substitutions in positions 2,4 of the porphyrin ring. The pH dependence of the association and dissociation of the deuteroheme-RHx complex showed 50% saturation at pH 4.9 ± 0.1 and 10.7 ± 0.1. At pH values from 6 to 9.5, binding was equimolar, whereas none occurred below pH 4.0 or above pH 11.2. The circular dichroism spectrum of RHx exhibits a prominent positive maximum at 231 nm, which is tentatively ascribed to tryptophan. The ellipticity at 231 nm increases approximately 50% upon formation of the deuteroheme-RHx complex. This band is abolished both by N-bromosuccinimide treatment and by lowering the pH to 2 or raising it to 11.