Iron Protoporphyrin Research Articles

Chemical and transient state kinetic studies on the formation and decomposition of horseradish peroxidase compounds XI and XII.

Previous studies on the chlorination reaction catalyzed by horseradish peroxidase using chlorite as the source of chlorine detected the formation of a chlorinating intermediate that was termed Compound X (Shahangian, S., and Hager, L.P. (1982) J. Biol. Chem. 257, 11529-11533). These studies indicated that at pH 10.7, the optical absorption spectrum of Compound X was similar to the spectrum of horseradish peroxidase Compound II. Compound X was shown to be quite stable at alkaline pH values. This study was undertaken to examine the relationship between the oxidation state of the iron protoporphyrin IX heme prosthetic group in Compound X and the chemistry of the halogenating intermediate. The experimental results show that the optical absorption properties and the oxidation state of the heme prosthetic group in horseradish peroxidase are not directly related to the presence of the activated chlorine atom in the intermediate. The oxyferryl porphyrin heme group in alkaline Compound X can be reduced to a ferric heme species that still retains the activated chlorine atom. Furthermore, the reaction of chlorite with horseradish peroxidase at acidic pH leads to the secondary formation of a green intermediate that has the spectral properties of horseradish peroxidase Compound I (Theorell, H. (1941) Enzymologia 10, 250-252). The green intermediate also retains the activated chlorine atom. By analogy to peroxidase Compound I chemistry, the heme prosthetic group in the green chlorinating intermediate must be an oxyferryl porphyrin pi-cation radical species (Roberts, J. E., Hoffman, B. M., Rutter, R. J., and Hager, L. P. (1981) J. Am. Chem. Soc. 103, 7654-7656). To be consistent with traditional peroxidase nomenclature, the red alkaline form of Compound X has been renamed Compound XII, and the green acidic form has been named Compound XI. The transfer of chlorine from the chlorinating intermediate to an acceptor molecule follows an electrophilic (rather than a free radical) path. A mechanism for the reaction is proposed in which the activated chlorine atom is bonded to a heteroatom on an active-site amino acid side chain. Transient state kinetic studies show that the initial intermediate, Compound XII, is formed in a very fast reaction. The second-order rate constant for the formation of Compound XII is approximately 1.1 x 10(7) M-1 s-1. The rate of formation of Compound XII is strongly pH-dependent. At pH 9, the second-order rate constant for the formation of Compound XII drops to 1.5 M-1 s-1. At acidic pH values, Compound XII undergoes a spontaneous first-order decay to yield Compound XI.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrochemical behaviour of hemin and PEO-hemin in ion-conductive polymer matrices

Both iron(III) protoporphyrin IX (hemin) and polyethylene oxide (PEO)-modified hemin (PEO-hemin) were incorporated into liquid or solid state ion-conductive polymer matrices. Their electrochemical behaviour was examined by cyclic voltammetry. Under nitrogen, PEO-hemin (like hemin) shows two redox processes in PEO oligomer. However, in the PEO solution of hemin, only one electrocatalytic reduction wave of dioxygen was observed at −0.50 V (vs. Ag). PEO-hemin also catalysed the electrochemical reduction of dioxygen in solution, and two waves appeared at −0.50 and −0.65 V (vs. Ag). Hemin cannot be dispersed well in solid polymer electrolytes because of the affinity problem. On the contrary, PEO-hemin can be dispersed into them at the molecular level. Similar redox behaviour of PEO-hemin was detected in solid polymer electrolytes. In the solid state, the redox potential of PEO-hemin is always higher than that in solution.

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)

Determining the cause of anemia

In the vast majority of cases, the cause of microcytic hypochromic anemia is clearly suggested by the patient history, physical examination results, red cell indexes, and peripheral blood smear. Thus, further diagnostic testing, if necessary, can be very selective. When the underlying cause of anemia is obscure, the serum ferritin concentration should be measured first. If it is normal or increased, serum iron and free erythrocyte protoporphyrin levels can be determined. The serum iron level is low in anemias caused by iron deficiency and chronic disease but normal or elevated in those resulting from the thalassemias, hemoglobin E disorders, and lead toxicity. The free erythrocyte protoporphyrin level is elevated with iron deficiency, the anemia of chronic disease, and lead toxicity but normal with thalassemias and hemoglobin E disorders. Results of these two test indicate which of the more specific tests is most likely to yield the correct diagnosis.

Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding.

Prostaglandin endoperoxide (PGH) synthase has a single iron protoporphyrin IX which is required for both the cyclooxygenase and peroxidase activities of the enzyme. At room temperature, the heme iron is coordinated at the axial position by an imidazole, and about 20% of the heme iron is coordinated at the distal position by an imidazole. We have used site-directed mutagenesis to investigate which histidine residues are involved in PGH synthase catalysis and heme binding. Individual mutant cDNAs for ovine PGH synthases were prepared with amino acid substitutions at each of 13 conserved histidines. cos-1 cells were transfected with each of these cDNAs, and the cyclooxygenase and peroxidase activities of the resulting microsomal PGH synthases were measured. Mutant PGH synthases in which His-207, His-309, or His-388 was replaced with either glutamine or alanine lacked both activities. Gln-386 and Ala-386 PGH synthase mutants exhibited cyclooxygenase but not peroxidase activities. Other mutants exhibited both activities at varying levels. Because binding of heme renders native PGh synthase resistant to cleavage by trypsin, we examined the effects of heme on the relative sensitivities of native, Ala-204, Ala-207, Ala-309, Ala-386, and Ala-388 mutant PGH synthases to trypsin as a measure of the heme-protein interaction. The Ala-309 PGh synthase mutant was notably hypersensitive to tryptic cleavage, even in the presence of exogenous heme; in contrast, the native enzyme and the other alanine mutants exhibited similar, lower sensitivities toward trypsin and, except for the Ala-386 mutant, were partially protected from trypsin cleavage by heme. Preincubation of the native and each of the alanine mutant PGH synthases, including the Ala-309 mutant, with indomethacin protected the proteins from trypsin cleavage. Thus, all the mutant proteins retain sufficient three-dimensional structure to bind cyclooxygenase inhibitors. Our results suggest that His-309 is one of the heme ligands, probably the axial ligand, of PGH synthase. Two other histidines, His-207 and His-388, are essential for both PGH synthase activities suggesting that either His-207 or His-388 can serve as the distal heme ligand; however, the trypsin cleavage measurements imply that neither His-207 nor His-388 is required for heme binding. This is consistent with the fact that only 20% of the distal coordination position of the heme iron of PGH synthase is occupied by an imidazole side chain.

Perspectives In Aquatic Toxicology

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Comparison of Hemin Enhancement of Burst-Forming Units-Erythroid Clonal Efficiency by Progenitor Cells from Normal and HIV-Infected Patients

The ability of peripheral-blood hematopoietic progenitor cells from AIDS patients and normal controls to respond to erythropoietin (Epo) was assessed for burst-forming units-erythroid (BFU-E). BFU-E colony formation from AIDS patients' peripheral blood responded to a wide range of Epo concentrations (0.5-4 U) in a similar manner as erythroid progenitors obtained from normal peripheral blood. The optimum dose response of BFU-E to Epo was 2 U which resulted in generation of 71 +/- 4 BFU-E in AIDS patients (n = 10), as compared to 77 +/- 5 BFU-E in normal donors (n = 3). The optimum concentration range of hemin enhancement of erythroid progenitor BFU-E was 10-50 microM. In all instances, Epo was essential for BFU-E growth. Inclusion of hemin at a concentration of 10 microM in AIDS patients' peripheral-blood erythroid progenitor cells resulted in enhancement of BFU-E by 136-215%. Similarly, inclusion of hemin (10-100 microM) in normal bone marrow erythroid progenitor cell cultures resulted in enhancement of BFU-E. Inclusion of an equivalent amount of iron or tin protoporphyrin to progenitors cells from AIDS patients' peripheral blood had no effect on the number of colonies observed. On the other hand, inclusion of another heme analogue, zinc protoporphyrin, in AIDS or normal cultures resulted in a 50% suppression of BFU-E colony formation. These results demonstrate that peripheral-blood mononuclear cells from AIDS patients retain the capacity to generate erythroid precursors such as BFU-E in the presence of Epo, and that hemin has a specific enhancement effect on growth of BFU-E colony formation obtained from peripheral blood or bone marrow cells.

Study on the electrocatalytic reduction of H 2O 2 at iron protoporphyrin modified electrode with a rapid rotation-scan method

The electrochemical behaviour for H 2O 2 reduction at iron protoporphyrin modified pyrolytic graphite (FePP/PG) electrode has been studied by cyclic voltammetry and rotating disc electrode ( rde). The peak potential ( E p) of the catalytic wave at FePP/PG electrode in the presence of H 2O 3 is more positive than that ( E 1 2 ) of the adsorption wave at the same modified electrode in the absence of H 2O 2. The electrocatalytic kinetics for H 2O 2 reduction at rotating FePP/PG disc electrode was investigated with a rapid rotation-scan method, and the rate constants ( k) at various pH values and potentials were measured. A regular variation of k values with the electric charges ( Q) passed through FePP/PG electrode during the catalytic reduction of H 2O 2 was observed. If disc potential is negative enough, the catalytic reaction is controlled by a diffusion process. At more positive potentials it is controlled by a kinetic process. The electrocatalytic stability of the modified electrode increases with increasing solution pH, and the decrease of k with Q values becomes slower.

Photochemically modified myeloperoxidase, with optical spectral properties analogous to those of lactoperoxidase, retains its original catalytic activity

During the course of a reducing reaction using ketyl radicals generated from ketone photoreduction with ultraviolet light, a photoinduced chemical modification of the chromophore group in myeloperoxidase has been found. Light absorption and resonance Raman spectra for this modified enzyme indicated an iron porphyrin chromophore group. The alkaline pyridine hemochrome of the modified enzyme exhibited an optical spectrum closely related to that of iron protoporphyrin IX. The chromophore group of the modified myeloperoxidase was cleaved from the protein by methoxide. Proton magnetic resonance of the diamagnetic bis(cyanide) compound of the extracted heme group showed the presence of two vinyl and three methyl side chains associated with a porphyrin macrocycle. These data provide further insight into the structure of the active site in myeloperoxidase. The EPR spectral properties and enzymatic activities of the native myeloperoxidase are essentially conserved in the modified enzyme. Our present results indicate that the heme peripheral substituent is modified while the stereochemical structure surrounding the chromophore group is not altered by the photochemical modification.

Mechanisms of Organophosphorus Ester-Induced Delayed Neurotoxicity: Type I and Type II

Some organophosphorus compounds produce neurologic dysfunctions, known as OPIDN, after a delay period that is accompanied by neuropathic damage in the central and peripheral nervous systems. This group of chemicals may be divided into two classes, Type I and II, based on chemical structure, species selectivity, age sensitivity, the length of latent period, clinical signs, morphology and distribution of neuropathologic lesions, protection with phenylmethyl sulfonyl fluoride, inhibition of neurotoxic esterase, and effect on catecholamine secretion from bovine adrenome-dullary chromaffin cells. The importance of this effect is underlined by the fact that incidents involving more than 40,000 cases of OPIDN in humans have been documented from 1899 to 1989. Most of these compounds are direct or indirect inhibitors of AChE, and produce acute cholinergic effects. Neurologic deficits are characterized by three phases: progressive, stationary, and improvement. Prognosis of OPIDN depends on the extent of damage of the nervous system. Improvement or even recovery of functions may follow mild cases, whereas severe toxicity results in long-lasting neurologic dysfunctions reflecting spinal cord damage. Recent studies have shown that delayed neurotoxic organophosphorus compounds interact with Ca2+/calmodulin kinase II (CaM kinase II), an enzyme responsible for the endogenous phosphorylation of cytoskeletal proteins, i.e. microtubules, neurofilaments, and MAP-2. This leads to an increased activity of CaM kinase II and enhanced phosphorylation of cytoskeletal elements, and eventually in the disassembly of cytoskeletal proteins. The dissociation of cytoskeletal proteins causes increased fast axonal transport in the treated animals resulting in the accumulation of altered cytoskeletal elements in the distal portions of the axon. Abnormal tubulin and neurofilaments are transformed into filamentous polymers and undergo condensation and dissolution. Concomitantly, proliferated endoplasmic reticulum and accumulated mitochondria degenerate and release Ca2+ ions. This leads to Ca2(+)-activated proteolysis of the cytoskeleton and interruption of ionic balance across the axonal membrane resulting in the uptake of water and axonal swelling, which subsequently degenerates. A similar mechanism may cause secondary myelin degeneration.

The electrochemistry of hemin in dimethylsulfoxide

The electrochemistry of iron(III) protoporphyrin IX chloride (hemin) in dimethylsulfoxide (DMSO) was examined under both aerobic and anaerobic conditions. Under anaerobic conditions, two redox processes were observed ▪, and a diffusion coefficient of 1.3 × 10 −6 cm 2 s was calculated for the iron(III) form of hemin. In the presence of oxygen, new peaks were observed, in particular a peak at −0.40 V evidently due to reduction of an oxygen adduct of hemin. This process occurs at a potential of about 0.4 V less negative than the reduction of oxygen in the absence of hemin. Spectroelectrochemistry, hydrodynamic voltammetry and chronocoulometry were used in conjunction with cyclic voltammetry to characterize the hemin redox system. Possible application of the system to the development of a porphyrin based oxygen sensor is discussed.

The Interaction Of The Beta-Haloethyl Benzylamines, Xylamine, And DSP-4 With Catecholaminergic Neurons

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Low-spin iron(III) porphyrins encapsulated in aqueous detergent micelles: proton- and nitrogen-15 nuclear magnetic resonance studies

Variable temperature 1H and 15N n.m.r. studies have been made on low-spin iron(III) protoporphyrin IX (3,7,12,17-tetramethyl-8,13-divinylporphyrin-2,18-dipropionic acid) complexes, [FeL(CN)2]– and [FeL(py)(CN)], encapsulated in aqueous detergent micelles. The haem is shown to be monodispersed in the micellar solution, which ensures the paramagnetic shift is free from the effect of aggregation. The effects of the hydrophobic interactions of the micellar cavity on the linewidth, and the magnitude and spread of the chemical shift, are discussed and compared with those in absence of micelles and in haemoproteins. The temperature dependence of the paramagnetic shift of the haem methyl proton shows deviation from Curie's law, which is discussed in terms of the electronic structure of haem. The effect of the size and hydrophobicity of the micelles is further shown in the 15N n.m.r. signal of the bound CN–, where the downfield shift follows the trend: absence of detergent < sodium dodecyl sulphate < Triton X-100 < hexadecyltrimethylammonium bromide.

Role Of Pharmacokinetics In Safety Evaluation And Regulatory Considerations

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

A six-coordinate high spin protoporphyrin IX iron(III) complex as a model for ferric haemproteins: Mössbauer spectrum of bisaquo(protoporphyrinato IX)iron(III) encapsulated in aqueous detergent micelles

The Mössbauer spectrum is reported for a six- coordinate high spin iron(III) protoporphyrin complex, [(PPIX)Fe(H 2O) 2] +, which is monodispersed in an aqueous frozen solution of sodium dodecyl sulphate micelles. The isomer shift of 0.46 mm −1 and the quadrupole splitting of 1.19 mm 1̄ are similar to those of aquo met myoglobin. These results are compared with the literature results on other high spin six-coordinated models and with those of some proteins. This is the first report of a Mössbauer study on a haem encapsulated in a detergent micelle.

Animal Models For The Study Of Drugs In Ischemic Stroke

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Percutaneous Absorption Of Drugs

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Review of Reviews

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Drug nephrotoxicity.

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Molecular Pharmacologic Approaches To The Treatment Of Aids

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More