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The interaction of human serum albumin and hemopexin with porphyrins

1980; Elsevier BV; Volume: 624; Issue: 1 Linguagem: Inglês

10.1016/0005-2795(80)90246-9

1878-1454

William T. Morgan, Ann Smith, P Koskelo,

Porphyrin Metabolism and Disorders

The interactions of the porphyrin-binding proteins of human serum, albumin and hemopexin, with coproporphyrin I and III, uroporphyrin I, protoporphyrin IX and zinc-protoporphyrin IX were examined quantitatively by means of equilibrium dialysis and fluorescence. Albumin bound the porphyrins with a high affinity, dissociation constant (Kd) in the μM range, with the exception of uroporphyrin I which was only weakly bound, Kd of the order of 100 μM. Protoporphyrin IX and zinc-protoporphyrin IX were more strongly bound than coproporphyrin I and III suggesting common binding sites. Human hemopexin formed complexes with coproporphyrin I and III, Kd near 0.5 μM, with protoporphyrin IX, Kd near 2 μM, and with zinc-protoporphyrin IX, Kd near 0.5 μM. All porphyrins were bound with an apparent stoichiometry of 1 : 1, except zinc-protoporphyrin, 2 mol of which appear to be bound per mol of hemopexin. As with albumin, only a weak interaction with uroporphyrin I is evident, Kd near 50 μM. Formation of the heme-hemopexin complex almost completely abolished subsequent interaction with coproporphyrin, consistent with heme and porphyrins sharing the same primary binding site on hemopexin. Several types of competition experiment were conducted to test directly the relative affinities of the two proteins for these porphyrins. Under these conditions, albumin clearly has a higher affinity than hemopexin for both protoporphyrin and zinc-protoporphyrin. Hemopexin is able to compete effectively with albumin for coproporphyrin I or III only at low molar ratios of the two proteins and seems unlikely to bind a significant portion of these porphyrins at the molar ratios (about 1 : 40) of hemopexin and albumin found in human serum. Further evidence for species differences in the heme binding site of hemopexin was obtained from fluorescence measurements on the human and rabbit proteins. Firstly, human hemopexin displayed a higher apparent stoichiometry of zinc-protoporphyrin binding (1.9 : 1) than rabbit hemopexin (1.3 : 1), with zinc-protoporphyrin-human hemopexin exhibiting a slightly higher relative fluorescence at 595 nm. Secondly, protoporphyrin bound to human hemopexin had red-shifted excitation (410 vs. 407 nm) and emission (634 vs. 627 nm) maxima compared with rabbit hemopexin. Thirdly, coproporphyrin I produced little quenching of the human protein compared with that of the rabbit protein.