Interaction of rabbit hemoplexin with copro- and uroporphyrins.

Rabbit hemopexin forms equimolar complexes in vitro with the I and III isomers of both coproporphyrin and uroporphyrin. The apparent dissociation constants (Kd) of these complexes are estimated to be 4-10(-7) M for coproporphyrin-hemopexin and 10(-6) M for uroporphyrin-hemopexin by equilibrium dialysis and quenching of protein fluorescence. Results of competitive binding experiments suggest that all four porphyrins bind at the heme-binding site of hemopexin, and that the relative affinity of rabbit hemopexin for these porphyrins is: deuteroheme greater than coproporphyrin I or III greater than uroporphyrin I or III. These findings provide further evidence that hemopexin may function as a transport protein for circulating coproporphyrins as well as for heme.

Transfer of heme from heme-albumin to hemopexin.

Exchange of heme in vitro between two heme-binding serum proteins, albumin and hemopexin, was examined spectrophotometrically. Hemopexin, albumin and heme in molar ratios of 1 : 70 : 1 were incubated at 22 degrees C, pH 7.3. The heme was added as free heme, heme-hemopexin or methemalbumin. Due to the high affinity of hemopexin for heme, Kd near 10(-13) M, only negligible amounts of heme were transferred from hemopexin to albumin in 48 h. However, more than 80% of heme was transferred from methemalbumin to hemopexin within 24 h. Heme added to a 1 : 70 mixture of the apo-proteins is initially bound by albumin; but more than 90% is bound by hemopexin in 24 h. Addition of dithionite causes nearly all of the heme present, whether added as free heme or methemalbumin, to associate with hemopexin in 15 min. Albumin thus appears to have a much lower affinity for ferro- than for ferri-heme. Results obtained from similar experiments with human serum and human serum made hemopexin-free by immunoadsorption fully corroborate those obtained with mixtures of purified albumin and hemopexin. These observations suggest that the rate-limiting step in the heme transport function of hemopexin is the formation of the heme-hemopexin complex, rather than the uptake of the complex by the liver.

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.