A continuous anaerobic fluorimetric assay for ferrochelatase by monitoring porphyrin disappearance
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Keywords:
Ferrochelatase
Protoporphyrin IX
Tetrapyrrole
Hemin
Administering acid (ALA) to isolated pea (Pisum sativum L.) chloroplasts resulted in an increase of heme synthesis in the heme branch of the tetrapyrrole pathway. At 0.1 mM ALA, in the presence of 1 mM heme synthesis was stimulated up to 7 fold of that in the absence of . N-Methylmesoporphyrin IX (NMMP), a powerful inhibitor of ferrochelatase, inhibited heme synthesis by 95% at one micromolar concentration. The addition of A TP to the chloroplasts caused not only heme synthesis, but Mg-protoporphyrin IX synthesis in the chlorophyll branch of the tetrapyrrole pathway. In the presence of NMMP, however, inhibition of Mg-protoporphyrin IX synthesis was not observed whereas heme synthesis was inhibited completely.
Tetrapyrrole
Ferrochelatase
Protoporphyrin IX
Hemeprotein
Protochlorophyllide
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Abstract 5‐Aminolevulinic acid (5‐ALA) is a natural precursor of protoporphyrin IX (PpIX), which can be used as a photosensitizer in photodynamic therapy (PDT). Accumulation of PpIX in benign meningioma cells has been observed previously, its exploitation for PDT, however, was discouraged by inconsistent results. To evaluate PDT for benign meningiomas, we investigated PpIX synthesis in two human meningioma cell lines (HBL‐52 and BEN‐MEN‐1), their respective extracellular loss of PpIX and corresponding ferrochelatase (FECH) activity as well as their susceptibility to PDT. We demonstrated PpIX production after 5‐ALA administration and minor loss to the extracellular space in both cell lines. However, significantly more (up to five times) PpIX was accumulated in BEN‐MEN‐1 as compared with HBL‐52 cells. FECH activity was 2.7‐fold higher in HBL‐52 compared with BEN‐MEN‐1 cells and accordingly higher FECH levels could be shown in HBL‐52 cells by Western blot analysis. BEN‐MEN‐1 cells were much more sensitive to PDT and cells could be almost completely killed by irradiation doses of 2 J cm −2 , whereas HBL‐52 showed only an insufficient response at this irradiation dose. We conclude that differences in intracellular PpIX concentrations between HBL‐52 and BEN‐MEN‐1 benign meningioma cells were mainly due to differences in FECH activity and that these differences correspond to their susceptibility to 5‐ALA‐induced PDT.
Ferrochelatase
Protoporphyrin IX
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Protoporphyrin (IX) and its dimethyl ester were obtained in homogeneous polycrystals of high purity, in a good yield, from hemin by the modification of Ramsey's method. Purity and physicochemical properties of these crystals were confirmed by ultraviolet, infrared, and NMR spectra, differential thermal analysis, and X-ray power diffraction analysis. The dimethyl ester had hitherto been reported as melting at 223∼230°but differential thermal analysis showed that a part of the molecular structure and crystal form underwent change accompanied by heat evolution and, therefore, this temperature was found to be the decomposition point. There was also an endothermic phenomenon at 180°, with transition of the crystal phase.
Hemin
Endothermic process
Protoporphyrin IX
Differential thermal analysis
Crystal (programming language)
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In vivo fluorescence studies of Cyanidium caldarium mutants grown in the dark in dextrose-containing media have shown that these organisms accumulate protoporphyrin IX. In the dark the accumulated protoporphyrin IX is gradually turned into a metalloporphyrin, Zn protoporphyrin. In the light, in the chlorophyll-lacking mutant GGB, both compounds are degraded and phycobiliproteins are formed. These results implicate protoporphyrin IX in situ as the general precursor to tetrapyrrole pigments and Zn protoporphyrin IX as a possible intermediate or regulator in the biosynthesis of phycobilins.
Protoporphyrin IX
Tetrapyrrole
Phycobiliprotein
Protochlorophyllide
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Ferrochelatase
Protoporphyrin IX
Porphobilinogen deaminase
Erythropoietic protoporphyria
3T3 cells
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Bovine skin fibroblasts accumulated protoporphyrin IX when incubated in culture with the porphyrin-heme precursor, delta-aminolevulinic acid (ALA). Fibroblasts from cattle homozygous for erythropoietic protoporphyria (EPP) and with the clinical symptoms of the disease accumulated approximately sixfold greater amounts of protoporphyrin IX than cells from normal control animals. Cells from obligatory heterozygous animals, which are clinically normal, accumulated an intermediate level of protoporphyrin IX. When these cells were incubated with ALA and CaMg EDTA, all types of cells accumulated approximately the same amount of protoporphyrin IX (approximately 500 nmol/mg protein), suggesting that ferrochelatase activity was equally low after inhibition by treatment with CaMg EDTA in all cells. Thus the ratio of protoporphyrin IX accumulation from ALA in cultures treated with CaMg EDTA compared with controls treated with ALA alone was greatest in normal cells, least in EPP cells, and intermediate in the heterozygote cells. These findings suggest that the amount of protoporphyrin IX accumulation from ALA reflects the extent of deficiency of ferrochelatase and is proportional to the dosage of abnormal EPP gene in cultured fibroblasts. Similarly, stimulation of porphyrin accumulation by CaMg EDTA reflects diminished ferrochelatase activity in these cells. Thus, the results of this study demonstrate the usefulness of estimating protoporphyrin IX formation from ALA for the detection of an EPP gene defect in cultured bovine skin fibroblasts.
Ferrochelatase
Erythropoietic protoporphyria
Protoporphyrin IX
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Ferrochelatase
Protoporphyrin IX
Tetrapyrrole
Hemin
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The common tetrapyrrole pathway of the photosynthetic bacterium, Rhodobacter capsulatus, ultimately leads to the synthesis of bacteriochlorophyll, heme, vitamin B-12, and siroheme. Molecular analysis of tetrapyrrole biosynthesis has been limited due to a lack of mutants. This study describes the isolation and characterization of a biotin-requiring mutant of R. capsulatus unable to synthesize heme. Growth conditions were established to allow selection and characterization of heme-deficient strains of R. capsulatus. A screening method was developed to isolate strains of R. capsulatus unable to synthesize heme. Tn5 derivatives of a bchB strain that exhibited an altered ability to synthesize the fluorescent porphyrin Mg-2,4-divinyl pheoporphyrin $a\sb5$ monomethyl ester were scored for hemin-dependence. One nonfluorescent, hemin-requiring mutant, strain AJB544, was isolated. This mutant could not grow on aminolevulinate or porphobilinogen whereas protoporphyrin and hemin supported growth. AJB544 was found to be a bio mutant, and it was shown that hemin could partially replace the requirement for biotin. Thin-layer chromatography and fluorimetric analysis revealed that AJB544 accumulated coproporphyrin and protoporphyrin when grown in minimal medium containing hemin or 2.5 nM biotin. Coproporphyrin was the major porphyrin that accumulated when AJB544 was grown in minimal medium containing hemin. Porphyrin formation by cell-free extracts of AJB544 and AJB478 demonstrated that AJB544 could form normal levels of protoporphyrin from protoporphyrinogen but not from aminolevulinate or porphobilinogen. Addition of biotin to extracts of AJB544 resulted in a 2.0 to 3.8-fold increase in protoporphyrin formation from aminolevulinate or prophobilinogen. Extracts of AJB544 grown in minimal medium containing biotin demonstrated an increase in protoporphyrin formation from aminolevulinate or porphobilinogen. These data suggest that biotin is required for the conversion of coproporphyrinogen to protoporphyrinogen. Southern blot analysis demonstrated that strain AJB544 contained a single Tn5 insertion. AJB544 was characterized as a bioB mutant by crossfeeding studies and thin-layer chromatography. The bioB gene was cloned from R. capsulatus by screening a pLAFRI library of R. capsulatus DNA and localized to a 2.85 kb EcoRI-PstI fragment. Introduction of the bioB gene into AJB544 restored the fluorescent phenotype.
Hemin
Rhodobacter
Tetrapyrrole
Porphobilinogen
Protoporphyrin IX
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In previous studies it has been shown that reaction of crystalline horse spleen apoferritin with hemin leads to a protoporphyrin IX−apoferritin complex [Précigoux et al. (1994) Acta Crystallogr. D50, 739−743]. We show here the following. (i) Hemin binds to two classes of sites in horse spleen apoferritin at pH 8, each with a binding stoichiometry of 0.5 hemin/subunit; protoporphyrin IX also binds to horse spleen apoferritin with an apparent binding stoichiometry of 1 molecule of protoporphyrin IX/subunit. (ii) When Fe(III)-protoporphyrin IX binds to apoferritin, there is a pH-dependent loss of the metal ion, extremely slow at alkaline pH values (half-time of weeks) and much more rapid at acidic pH values (half-time of seconds below pH 5.0); maximum rates of demetallation are found at pH 4.0, and at lower pH values they decrease. (iii) Chemical modification of 11 carboxyl groups/subunit in horse spleen apoferritin does not affect hemin binding at alkaline pH values; however, it prevents hemin demetallation at acidic pH values. (iv) Hemin that has been demetallated at acidic pH values can be remetallated by increasing the pH; the rate of remetallation is greater at more alkaline pH values. (v) When around 20 atoms of iron/molecule are incorporated into horse spleen apoferritin and protoporphyrin IX is then bound, iron can subsequently be transferred to the porphyrin at pH 8.0. A mechanism is proposed to explain demetallation of heme, involving attack on the tetrapyrrole nitrogens of the protoporphyrin IX-Fe by protons derived from protein carboxylic acid groups and subsequent complexation of the iron by the corresponding carboxylates and binding of protoporphyrin IX to a preformed pocket in the inner surface of the apoferritin protein shell. The cluster of carboxylates involved is situated at the entrance to the pocket in which the protoporphyrin IX molecule is bound and has been previously identified as the site of iron incorporation into L-chain apoferritins. This appears to be the first example of iron removal and incorporation into porphyrins under relatively mild physiological conditions.
Hemin
Protoporphyrin IX
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Abstract It is shown that continuous illumination is mandatory for the induction of tetrapyrrole accumulation in acifluorfen‐sodium‐treated plants and for the photosensitization of tetrapyrrole‐dependent photodynamic damage. At low concentrations of acifluorfen‐sodium (up to 20 μM), photoporphyrin IX appears to be the major light‐induced tetrapyrrole that accumulates in the treated plants. At higher concentrations of acifluorfen‐sodium, monovinyl chlorophyllide a accumulates in addition to protoporphyrin IX. In the light, the development of photodynamic injury appears to be directly related to the accumulation of the light‐induced tetrapyrroles. For example when acifluorfen‐sodium‐treated plants are returned to darkness, or are treated with tetrapyrrole biosynthesis inhibitors, tetrapyrrole accumulation and photodynamic injury come to a halt. In‐vivo and in‐organello studies failed, however, to support the commonly held hypothesis that the induction of tetrapyrrole accumulation in the light, in acifluorfen‐sodium‐treated plants, is only dependent on the inhibition of protoporphyrinogen oxidase. Indeed, when plastids capable of very high rates of tetrapyrrole biosynthesis and accumulation were incubated with δ‐aminolevulinic acid and acifluorfen‐sodium, either in darkness or in the light, a severe inhibition of protoporphyrin IX and total terapyrrole formation was observed. Althoung these results are compatible with the inhibition of tetrapyrrole formation by acifluorfensodium at the level of protoporphyrinogen oxidase, they indicate that, in addition to that inhibition, other cuellular processes are probably involved in the light‐dependent accumulation of protoporphyrin IX in acifluorfensodium‐treated plants.
Tetrapyrrole
Protoporphyrin IX
Protoporphyrinogen oxidase
Protochlorophyllide
Photobiology
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