Heme Affinity Research Articles

Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs.

Heme oxygenase-2 (HO-2) degrades heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of heme regulatory motif (HRM) with a conserved core of Cys264-Pro265 and Cys281-Pro282. We examined interaction of HO-2 HRMs with Fe-PP, Zn-protoporphyrin IX (Zn-PP; HO-2 inhibitor), and protoporphyrin IX (PP IX). Spectral analyses, using 1:4 or 1:1 molar ratio of the heme to 10-residue peptides, corresponding to HRM containing HO-2 sequences, revealed specific interactions as indicated by a shift in the absorption spectrum of heme. Five residue peptides qualitatively produced similar results. Substitution of cysteine with alanine in either peptide eliminated interactions, and substitution of proline with alanine reduced the peptides' affinity for heme. Neither Zn-PP nor PP IX absorption spectrum was affected by HRM peptides. The circular dichroism spectra confirmed heme-HRM peptides interactions. An astounding 4,000-6,000-fold higher concentrations of KCN were required at pH 7.5 to displace HRM peptides from heme. Data suggest (a) each HRM can contribute to HO-2-heme interaction, (b) heme iron interacts with cysteine thiol, (c) charged residues upstream of Cys264-Pro265 result in its high-affinity heme binding, and (d) inhibition of HO-2 activity by synthetic metalloporphyrins does not involve HRMs. We suggest that heme bound to HRMs may serve as a binding site/reservoir for gaseous signal molecules.

Interaction of Cytochrome bd with Carbon Monoxide at Low and Room Temperatures: EVIDENCE THAT ONLY A SMALL FRACTION OF HEMEb595 REACTS WITH CO

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.

Structural and Dynamic Perturbations Induced by Heme Binding in Cytochrome b5,

The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.

Waterproofing the Heme Pocket: ROLE OF PROXIMAL AMINO ACID SIDE CHAINS IN PREVENTING HEMIN LOSS FROM MYOGLOBIN

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.

The Heme–Globin and Dimerization Equilibria of Recombinant Human Hemoglobins Carrying Site-Specific β Chains Mutations

The heme–globin and dimer–tetramer equilibria of ferric recombinant human hemoglobins with site-specific β chain mutations at the heme pocket or at either the α1β1 or the α1β2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the β chain heme and does not affect the dimer–tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of β chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the α chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of β chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, β chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.

Nitric oxide and vasospasm.

Delayed release of hemoglobin from the subarachnoid clot in the vicinity of conductive arteries leads to cerebral vasospasm after SAH. The mechanism of vasospasm however remains unclear. The one thousand times higher affinity of ferrous heme for nitric oxide, a potent vasodilator of cerebral vessels, than to oxygen, has led to the concept that hemoglobin scavenges nitric oxide and produces delayed cerebral vasospasm after SAH. For several years we have investigated the pathological mechanism(s) behind this event using in vivo experiments. We summarize experimental data and discuss the clinical value of the observations for the development of a new treatment for vasospasm after SAH.

From Myoglobin to Heme-Copper Oxidase: Design and Engineering of a CuB Center into Sperm Whale Myoglobin

Myoglobins (Mb) are small globular heme proteins that serve as O2 carriers. Heme-copper oxidases (HCOs) are large membrane-bound proteins that catalyze proton-coupled reduction of O2 to water. Mb contains a single heme center, while HCOs contain a high-spin heme-CuB dinuclear center, a low-spin heme center, and in certain subclasses of HCO enzymes such as cytochrome c oxidases (CcO) a dinuclear copper center called CuA. While Mb is one of the most well-characterized proteins, many questions about the structure and function of HCOs, such as the role of the CuB center, the origin of spin coupling between CuB and heme, and the exact nature of the reaction intermediates, remain to be fully understood. We report here the design and engineering of a copper-binding site in sperm whale myoglobin (swMb) based on structural comparison and computer modeling of swMb and CcO. UV−vis studies of the resting state of the designed protein swMb((L29H, F43H) (called CuBMb) suggest that a single copper-binding site is created in swMb. UV−vis, elemental analysis, and EPR studies of the cyanide-bound CuBMb indicate that a spin-coupled, CN--bridged CuB-heme center is formed in the designed model protein, as in the native HCOs. Parallel spectroscopic studies with Zn(II) in the place of Cu(II) further support the conclusion. The study also reveals that the presence of Cu(II) and Ag(I) (as a Cu(I) mimic) increased the affinity of heme for diatomic ligands such as CN- and O2. This study shows that it is possible to design and engineer metal-binding sites in proteins with little sequence and structural homology. The resulting designed protein, free from other chromophores, is more amendable to biochemical and biophysical studies. Spectroscopy studies of the designed protein indicate that the CuB center plays an important role in HCO structure and function.

Xanthones as antimalarial agents: stage specificity.

The erythrocytic development of Plasmodium falciparum is divided into the ring, trophozoite, and schizont stages based on morphologic assessment. Using highly synchronous ring and trophozoite cultures of P. falciparum, we observed considerable differences in their sensitivity to hydroxyxanthones: trophozoites were much more sensitive to the drugs than ring-stage parasites. Trophozoites treated with a prototypic xanthone, the 2,3,4,5,6-pentahydroxy derivative (X5), were arrested in their development and became degenerate in appearance within 24 hr of drug exposure. These morphologic changes appeared to reflect the cytotoxic nature of the action of the drug against the parasite, since daughter ring-stage forms were not observed following addition of the drug. That X5 was more active against parasites in the later stages of intraerythrocytic development is consistent with the proposed mode of action, inhibition of heme polymerization. Knowledge of the structure-activity relationships for xanthones as antimalarial agents has also been expanded. Xanthones with a hydroxyl group in the peri-position exhibited decreased antimalarial activity, possibly due to intramolecular hydrogen bonding with the carbonyl and consequent reduced affinity for heme. Paired hydroxyls attached to the lower half of the xanthone greatly enhanced drug potency.

Role of heme in structural organization of cytochrome c probed by semisynthesis.

The heme prosthetic group of cytochrome c is covalently attached to the protein through thioether bonds to two cysteine side chains. The role of covalent heme attachment to cytochrome c is not understood, and most heme proteins bind the prosthetic group by iron ion ligation and tertiary interactions only. A two-armed attachment seems redundant if the role of covalent connection is to limit heme group orientation or to decouple heme affinity from redox potential. These considerations suggested that one role for covalent attachment of the rigid planar heme might be in organizing the cytochrome c protein structure. Indeed, porphyrin cytochrome c (in which the heme iron ion has been removed) is substantially more ordered than apocytochrome c, having characteristics consistent with a molten globule state. To assess the importance of planar rigidity in ordering this protein, semisynthesis was used to substitute porphyrin by two hydrophobic surrogates, one based on biphenyl and the other on phenanthrene, which have different degrees of planarity and rigidity. The expected two-armed covalent attachment of each surrogate was confirmed in the protein products by a variety of methods including mass spectrometry and NMR. Despite being only about half the size of the porphyrin macrocycle, and lacking any possibility for ligation or polar group interactions with the surrounding protein, the two surrogates confer helix contents that are comparable to that of the molten globule formed by porphyrin cytochrome c under similar solution conditions. The pH titrations of the derivatives monitored by circular dichroism exhibit reversible, bell-shaped folding and unfolding transitions, implying that charge group interactions in the protein are involved in stabilizing the helical structures formed. The thermal transitions of the two derivatives at neutral pH are cooperative, with similar midpoints. The similarity of helical content and structural stability in the two derivatives indicates that the increase in conformational freedom by the biphenyl surrogate does not substantially reduce protein structural stability. The similarity of the two derivatives to porphyrin cytochrome c suggests that the common feature among the three covalently attached groups-their hydrophobicity-is by far the dominant factor in organizing stable structures in the protein.

Histidine placement in de novo-designed heme proteins.

The effects of histidine residue placement in a de novo-designed four-alpha-helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [[Ac-CGGGELWKL x HEELLKK x FEELLKL x HEERLKK x L-CONH2]2]2 composed of a generic (alpha-SS-alpha)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [[Ac-CGGGELWKL x AEELLKK x FEELLKL x AEERLKK x L-CONH2]2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis-histidine site formed between heptad positions a at 10 and 10' bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 1.5 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 microM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 microM. The bis-histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.

Heme as a signaling molecule under environmental stress

One of the most remarkable revolutions during the history of animal kingdom is the adaptation to oxygen, a highly toxic gas produced by plants. Based on the high affinity of heme toward oxygen, it has been believed that heme itself and/or hemoprotein should play significant roles in the processes of adaptation. Recently, two novel functions of heme and its metabolites were identified; namely, hemoprotein is an oxygen sensor and biliverdin and bilirubin, catabolites of heme, are antioxidants. Thus, heme is a key molecule in the responses to environmental stress, including oxygen. Although activation of hypoxia inducible factor-1, which induces expression of genes encoding erythropoietin and heme oxygenase (HO-1), is generally accepted to be controlled by oxygen sensor, the precise signaling pathway has not yet been well elucidated. Various stresses such as hypoxia are known to activate the HO-1 gene, the key enzyme of heme degradation, resulting in the marked conversion of pro-oxidant (heme) into its antioxidative catabolites. The induction is, therefore, supposed to have protective effects. Recent reports on HO-1 deficiency also support this hypothesis that the activation of the HO-1 gene is one of the most important defense mechanisms against environmental stress.

Hemopexin Decreases Spontaneous Chemiluminescence of Cold Preserved Liver after Reperfusion

Hemopexin is a plasma protein with exceptionally high affinity for heme. During liver transplantation heme is released via lysis of transfused blood. This heme may catalyze peroxidative reactions that contribute to “reperfusion” injury of the organ. Using a rat liver model of cold storage and reperfusion we tested the potential anti-oxidant effects of hemopexin. After 3 h of cold storage rat liver was reperfused with warm oxygenated buffer. Spontaneous liver chemiluminescence, which is a parameter of oxyradical production, was measured during reperfusion and expressed as an index of free radical production (IFRP). Chemiluminescence reached a maximum within 5 min of reperfusion and decreased to baseline within 30 min. Addition of hemopexin to the perfusate (5 μM) significantly decreased the IFRP. By contrast, the control proteins albumin and γ-globulin (10 μM) had a smaller non-significant effect. The data suggest that heme could be complexed by hemopexin during reperfusion, thus inhibiting heme mediated cellular injury.

Immobilized apo-myoglobin, a new stable reagent for measuring rates of heme dissociation from hemoglobin

Apo-myoglobin covalently linked on CNBr-activated Sepharose 4B is proposed as a new heme acceptor for investigating the heme transfer reaction from hemoproteins. Immobilized apo-myoglobin has the desirable properties of an ideal heme acceptor in that it is characterized by a high affinity for ferric heme, a high stability towards denaturation even at physiological temperatures and can be lyophilized for long-term storage. The study of heme release from myoglobin at pH 5.0 and 37°C indicates that heme affinity is increased at least 10-fold relative to the soluble protein. Experiments with human hemoglobin allowed the estimation of the heme release rates from both α and β chains and brought out the greater temperature sensitivity of the α chain heme-globin linkage.

Effect of four helix bundle topology on heme binding and redox properties.

We have designed two alternative four helix bundle protein scaffold topologies for maquette construction to examine the effect of helix orientation on the heme binding and redox properties of our prototype heme protein maquette, (alpha-SS-alpha)2, previously described as H10H24 [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A. J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425]. Conversion of the disulfide-bridged di-alpha-helical monomer of (alpha-SS-alpha)2 into a single polypeptide chain results in topological reorientation of the helix dipoles and side chains within a 62 amino acid helix-loop-helix monomer, (alpha-l-alpha), which self-associates to form (alpha-l-alpha)2. Addition of an N-terminal cysteine residue to (alpha-l-alpha) with subsequent oxidation yields a 126 amino acid single molecule four helix bundle, (alpha-l-alpha-SS-alpha-l-alpha). Gel permeation chromatography demonstrated that (alpha-SS-alpha)2 and (alpha'-SS-alpha')2, a uniquely structured variant of the prototype, as well as (alpha-l-alpha)2 and (alpha'-l-alpha')2 assemble into distinct four helix bundles as designed, whereas (alpha-l-alpha-SS-alpha-l-alpha) elutes as a monomeric four alpha-helix bundle. Circular dichroism (CD) spectroscopy proves that these peptides are highly alpha-helical, and incorporation of four hemes has little effect on the helical content of the secondary structure. Four heme dissociation constants were evaluated by UV-visible spectroscopy and ranged from the 15 nM to 25 microM range for each of the peptides. The presence of Cotton effects in the visible CD illustrated that the hemes reside within the protein architecture. The equilibrium redox midpoint potentials (Em8) of the four bound hemes in each peptide are between -100 and -280 mV, as determined by redox potentiometry. The heme affinity and spectroelectrochemical properties of the hemes bound to (alpha-l-alpha)2 and (alpha-l-alpha-SS-alpha-l-alpha) are similar to those of the prototype, (alpha-SS-alpha)2, and to bis-histidine ligated b-type cytochromes, regardless of the global architectural changes imposed by these topological rearrangements. The hydrophobic cores of these peptides support local electrostatic fields which result in nativelike heme chromophore properties (spectroscopy, elevated reduction potentials, heme-heme charge interaction, and reactivity with exogenous diatomics) illustrating the utility of these non-native peptides in the study of metalloproteins.

A 65-kDa Protein Mediates the Positive Role of Heme in Regulating the Transcription of CYP2B1/B2 Gene in Rat Liver

Heme deficiency precipitated by CoCl2 administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and its transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl2 administration. The binding of nuclear proteins to labeled positive cis-acting element (-69 to -98 nucleotides) in the near 5'-upstream region of the gene is inhibited by CoCl2 administration to saline or PB-treated rats, as assessed in gel shift assays. Administration of exogenous hemin to the animal or addition in vitro to the extracts is able to overcome the effects of CoCl2 treatment. The protein mediating this effect has been purified from CoCl2 administered nuclear extracts by heparin-agarose, positive element oligonucleotide affinity, and heme affinity column chromatography. This 65-kDa protein manifests very little binding to the positive element, but in the presence of certain other nuclear proteins, shows a strong heme-responsive binding. The purified protein binds heme. It is also able to stimulate transcription of a minigene construct of the CYP2B1/B2 gene containing -179 nucleotides of the 5'-upstream region and the I exon in a cell-free system, manifesting heme response. It is concluded that the 65-kDa protein mediates the constitutive requirement of heme for the transcription of CYP2B1/B2 gene.

Ground and excited states of oxyheme: SAC/SAC-CI study

The SAC(symmetrie adapted cluster)/SAC-CI(configuration interaction) method is applied to the ground and low-lying excited states of oxyheme (FeC 23N 6O 2H 16). The ground state ( 1A′) is suitably represented by the Pauling model, Fe(II) (S = 0) + O 2( 1Δ g ) . The SAC-CI result reproduces well the lower excitation spectrum of oxyhemoglobin. The lowest peak observed at 1.34 eV is assigned to the 1Δ g → 1Σ g + excitation of the O 2 ligand. Many transitions originating from the iron d orbitals are calculated, although their intensities are very small. The lowest triplet state (1 3A″), which is due to the 1Δ g → 3Σ g − transition around the O 2 ligand, is calculated at 0.47 eV above the ground state, but its level is sensitive to the electron correlations included. In the geometry of the deoxy form, this 1 3A″ state becomes more stable than the closed-shell singlet state ( 1A′), indicating a geometrical control of oxygen affinity of heme.

The stability of holomyoglobin is determined by heme affinity.

The properties of wild-type, V68T, and H97D sperm whale myoglobins were compared to determine the relative importance of heme affinity and globin stability on the resistance of the holoprotein to denaturation. The V68T mutation decreases apoglobin stability by placing a polar side chain in the interior heme pocket. However, this substitution increases hemin affinity by formation of a strong hydrogen bond between coordinated water and the Thr68(E11) side chain. The H97D substitution disrupts favorable contacts with Ser92(F7) and the heme-7-propionate and causes a large increase in the rate of hemin dissociation. The Asp replacement has little affect on apoglobin stability because His97(FG3) is a surface residue. The aquomet, cyanomet, deoxyferrous, and apoglobin forms of each mutant and wild-type myoglobin were unfolded by titration with guanidinium chloride. Even though holomyoglobin denaturation involves the dissociation of heme and should be dependent on protein concentration, nonspecific heme binding to unfolded states makes the overall process appear to be a simple, unimolecular unfolding transition. The equilibrium constants for the denaturation of the holomyoglobin mutants correlate almost exclusively with heme affinity and not with the stability of the globin portion of the molecule. The strong correlation with heme affinity explains quantitatively why the stability of myoglobin is enhanced approximately 60-fold by reduction of iron to the ferrous deoxy state and by another approximately 100-fold with CO coordination. Parameters measured for GdmCl-, urea-, acid-, and heat-induced denaturation of holomyoglobins and hemoglobins reflect heme affinity and not the folding properties of the corresponding apoproteins. This conclusion suggests that (1) many previous studies of the denaturation of intact heme proteins need to be reevaluated in terms of heme affinity and (2) measurements with apoproteins are required for unambiguous determinations of the stability of globin structures.

The association rate constant for heme binding to globin is independent of protein structure.

Rate constants for CO-heme binding to 35 different recombinant apomyoglobins and several other apoproteins were measured in an effort to understand the factors governing heme affinity and the velocity of the association reaction. Surprisingly, the rate constant for the binding of monomeric heme is approximately 1 x 10(8) M-1 s-1 regardless of the structure or overall affinity of the apoprotein for iron-porphyrin. Major differences between the proteins are reflected primarily in the rates of dissociation of the prosthetic group. Slow phases observed in the reaction of CO heme with excess apomyoglobin result from formation of nonspecific heme-protein complexes which must dissociate before heme can bind specifically in the heme pocket. Once the specific heme-globin complex is formed, the heme pocket rapidly collapses around the porphyrin, simultaneously forming the bond between the proximal His93 and the heme iron atom. The overall affinity of sperm whale apomyoglobin for hemin is approximately 1 x 10(14) M-1. Nonspecific hydrophobic interactions between the porphyrin and the apolar heme cavity account for a factor of 10(5)-10(7). Covalent bond formation between Fe3+ and His93(F8) provides an additional factor of 10(3)-10(4). Specific interactions with conserved amino acids in the heme pocket contribute the final factor of 10(3)-10(4).

Expression of the mRNA of Heme-Binding Protein 23 Is Coordinated with That of Heme Oxygenase-1 by Heme and Heavy Metals in Primary Rat Hepatocytes and Hepatoma Cells

A 23-kDa protein with high affinity for heme (KD = 55 nM), therefore termed heme-binding protein 23 kDa (HBP23), was purified from rat liver cytosol [Iwahara, S., et al. (1995) Biochemistry 34, 13398-13406]. Homology search of the cloned HBP23 cDNA revealed that this protein belongs to a recently recognized class of thiol peroxidases, the antioxidant peroxiredoxin family. Since HBP23 gene expression was highest in the liver, HBP23 mRNA regulation by heme and heavy metals was investigated in cultures of primary rat hepatocytes and mouse hepatoma Hepa 1-6 cells. In both cell cultures HBP23 mRNA levels were upregulated in a time- and dose-dependent manner by heme. Heme-dependent induction of HBP23 mRNA occurred coordinately with that of the heme-metabolizing enzyme heme oxygenase-1, which was recently identified as inducible by oxidative stress. Treatment of primary rat hepatocyte or hepatoma cell cultures with the heavy metals CdCl2 (10 microM) and CoCl2 (300 microM) induced in parallel HBP23 and HO-1 mRNA levels, in the case of CdCl2 to even higher levels than heme. By contrast, mRNA expression of another heme binding protein, hemopexin, was not induced in hepatocyte cell cultures by heme or heavy metals. The data suggest that the expression of HBP23 and HO-1 mRNA is regulated by (a) similar mechanism(s) in liver and that both genes could play a common physiological role as antioxidants and/or in heme metabolism.

PREDICTION OF DRUG-DRUG INTERACTION CAUSED BY CYP3A INHIBITION: FROM IN VITRO TO IN VIVO, FROM ANIMAL TO HUMAN

Inhibitory effect of E4080 on the cytochrome P450 was studied using laboratory animals in vitro and in vivo.In vitro study E4080 has a high affinity for P450 heme in rat, dog, monkey and human liver microsomes. In rat microsomes, E4080 inhibited testosterone 6β and 16β-hydroxylase which are involved in CYP2B and CYP3A. Using human microsomes, E4080 inhibited metabolism of 7-benzyloxyresolfin, tolbutamide, bufuralol and testosterone. These results suggest that E4080 inhibits CYP 2B6, 2C9, 2D6 and 3A4 in human liver microsomes.In vivo study E4080 prolonged antipyrine half-life and decreased antipyrine clearance in rats, dogs and monkeys at the doses of 10mg/kg which exert pharmacological effect postulated from the minimal effective plasma concentrations(1-2 μg/ml ). The results may be suggested that E4080 interacts with antipyrine metabolism which is thought to be carried out by CYP 1A, 2C and 3A. In the clinical trial E4080 (100mg/man) decreased the excretion of urinary 6β-hydroxycortisol which is a marker of human hepatic cytochrome P4503A induction and inhibition.In conclusion, it is suspected that E4080 inhibits CYP3A not only in animals, but also in human and should be noticed drug-drug interaction in clinical use.