Heme Transfer Research Articles

Cytochrome P-450 heme moiety. The specific target in drug-induced heme alkylation.

Exogenously administered heme is incorporated into rat hepatic cytochrome P-450 in vivo (Correia, M. A., Farrell, G. C. Schmid, R. S., Ortiz de Montellano, P. R., Yost, G. S., and Mico, B. A. (1979) J. Biol. Chem. 254, 15-17). This was demonstrated in allylisopropylacetamide (AIA)-treated rats by the formation of a radioactive adduct derived from the porphyrin of the administered [3H]heme and AIA. Formation of such adducts requires catalytic participation of cytochrome P-450 in oxidative metabolism of AIA to an active species which subsequently alkylates the prosthetic heme moiety of the cytochrome. These results suggested that the exogenous heme had been incorporated prosthetically into cytochrome P-450 prior to generation of the adduct. However, the possibility remained that a minute portion of the inactivating AIA-species escaped the catalytic site of the generating hemoprotein and alkylated the nonprosthetically bound isotopic heme. To examine this critical possibility, we have employed a chemical derivative of heme which binds to the microsomal membrane. Although this heme derivative is a structurally suitable target for attack by the inactivating drug species, we found that it was unsuitable for incorporation into the prosthetic site of cytochrome P-450. The findings of this study provide irrefutable evidence that the label recovered in drug-porphyrin adducts is derived exclusively from radioactive heme incorporated prosthetically into cytochrome P-450. Drug-porphyrin adducts can therefore be used as reliable probes to follow the transfer of heme from the hepatic "free" heme pool into cytochrome P-450.

Effect of ligandin on the efflux of Co-deuteroporphyrin from isolated rat liver mitochondria

Summary Ligandin markedly accelerated the efflux of Co-deuteroporphyrin from tightly coupled mitochondria. This was associated with an increase in the rate of Co-deuteroporphyrin synthesis. The addition of 0.1 M KCl to the medium reduced but did not abolish the effect on efflux. In the absence of Co 2+ , ligandin inhibited deuteroporphyrin uptake by mitochondria. When added to sonicated mitochondria ligandin inhibited Co-deuteroporphyrin synthesis. These results suggest that ligandin plays an important part in the transfer of heme from mitochondria to its other sites of utilization. The effect of ligandin on heme release requires intact functional mitochondrial membranes and overcomes the tendency to deplete the porphyrin substrate for which it has affinity.

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°C, pH 7.3. The heme was added as free heme, heme-hemopexin or methemalbumin. Due to the high affinity of hemopexin for heme, K d 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.

Non-equivalence of human hemoglobin chains in the oxidation-reduction and heme-transfer reactions. A 13C nuclear-Magnetic-resonance study.

12C nuclear magnetic resonance (NMR) spectroscopy has been applied to the investigation of chain non-equivalence for two reactions of human hemoglobin: oxidation-reduction and hemetransfer. The method is based on previous observations that in the carbonyl region, Hb13CO gives two well-resolved resonances which arise from 13C of carbonyls bound respectively to the alpha and beta chains; moreover, integration of spectra allows on e to estimate their relative abundance. A mixture of ferrous and ferric hemoglobins in dye-mediated oxidation-reduction equilibrium can be formally considered to be equivalent to two redox couples in equilibrium, namely alphaIII/alphaII and betaIII/betaII; from a knowledge of these ratios, one can conclude whether the chains are equivalent or not in their oxidation-reduction properties. In this work, these ratios were evaluated by reacting the redox systems with 13CO and integrating the 13C NMR spectra. The results show differences in the intrinsic oxidation-reduction potentials of the chains in hemoglobin tetramer, E1/2(beta)being higher than E1/2(alpha)in neutral solution but not at pH9 and above. The binding of inositol hexakisphosphate does not modify the difference between beta and alpha though substantially increasing the overall potential The results are discussed in the light of current hypotheses to account for the change of Hill coefficient with pH for the reaction studied. The non-equivalence of chains is shown also for heme transfer from methemoglobin. For the phosphate-free protein, the beta chains lose heme more rapidly than that alpha chains; the addition of inositol hexakisphosphate results in the decrease of overall heme transfer as well as of chain heterogeneity.

Transfer of heme from ferrihemoglobin and ferrihemoglobin isolated chains to hemopexin.

Incubation of ferrihemoglobin as well as of hemoglobin isolated chains in ferri‐form with hemopexin at pH 7.0 leads to the gradual transfer of heme to the hemopexin molecules until the equilibrium is attained.The reaction of ferrihemoglobin with hemopexin can be described as a four‐stage process involving successive transfer of the four heme groups from hemoglobin tetramer to the four molecules of hemopexin. The heme binding sites of the hemoglobin molecule are nonequivalent and cooperative.The transfer of heme from the isolated hemoglobin chains in ferri‐form occurs as the simple competitive reaction which can be characterized by a single equilibrium constant.The affinity constant value for the binding of heme to the single binding center of hemopexin molecule was estimated as 1.9 × 1014× M−1.The direct transfer of heme from hemoglobin to hemopexin may occur in vivo as a part of hemoglobin degradation process.

Transfer of heme from mitochondria in rat liver cells.

The final steps in heme synthesis take place within mitochondria while the acceptor apoproteins are synthesized on the endoplasmic reticulum. When 14C-δ-aminolevulinic acid is used as a heme precursor in intact rats, measurable 14C-heme is found to be associated with the microsomes within 10 min of intraperitoneal injection. This rapid transfer of heme from mitochondria was studied in vitro using isolated rat liver mitochondria, and protoporphyrin IX and 59Fe as heme precursors. These mitochondria synthesize heme when suspended in whole cell sap and this is only partially reduced by substituting Sephadex G-25 filtered cell sap or sucrose. Mitochondria incubated in G-25 filtered cell sap or sucrose synthesize equivalent amounts of heme but those in sucrose export little heme into the surrounding medium. Heme export from mitochondria is dependent on protein in the suspending medium. In cell sap, heme is associated with multiple proteins and no single carrier was identified. Heme probably makes its way from mitochondria to microsomes via various protein carriers by nonspecific adherence. Microsomal apoproteins or other heme binding proteins then remove the heme from the intermediate carrier as the terminal step.

Cytochrome c3. A class of electron transfer heme proteins found in both photosynthetic and sulfate-reducing bacteria

Cytochromes c with absorption spectra and oxidation-reduction potentials very similar to those of Desulfovibrio vulgaris cytochrome c 3 were isolated from the photosynthetic bacteria Chloropseudomonas ethylicum, Rhodopseudomonas spheroides and Rps. palustris, as well as from the blue-green alga Anacystis nidulans. Analysis of the highly purified C. ethylicum cytochrome c-551.5 indicates that this protein contains three heme groups per molecule, as compared to Desulfovibrio cytochrome c 3, which has four hemes. It is suggested that a special class of cytochromes c be recognized which has absorption spectra and oxidation-reduction potentials similar to those of Desulfovibrio cytochrome c 3.

Heme transfer from hemoglobin and ferrihemoglobin to some new ligands and its implication in the mechanism of oxidation of ferrohemoglobin by air

Heme transfer from hemoglobin and ferrihemoglobin to some new ligands and its implication in the mechanism of oxidation of ferrohemoglobin by air

Heme transfer from fetal hemoglobin in the presence of sodium hydrosulfite

Heme transfer from fetal hemoglobin in the presence of sodium hydrosulfite