The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

Photosensitized formation of singlet oxygen in water solutions of covalent porphyrin-antibody conjugates

The effective photogeneration of singlet molecular oxygen (1O2) by porphyrins (coproporphyrin I; 2,4-bi (alpha-methoxyethyl) deuteroporphyrin IX and cyclopanten-coproporphyrin I) conjugated with antibodies (mouse monoclonal IgG and IgM and human gamma-globulin) have been observed with the direct luminescence method of 1O2 detection. Absolute quantum yields of 1O2 formation by the conjugates have been determined. The data suggest that porphyrin-antibody conjugates are promising for the use as drugs in photodynamic tumor treatment.     

Inhibitory effect of porphyrins on the proliferation of mouse spleen lymphocytes in vitro

The influence of various porphyrins (deuteroporphyrin IX, mesoporphyrin IX, protoporphyrin IX, hematoporphyrin) and two related compounds (hemin, biliverdin) on the spontaneous proliferation of mouse spleen lymphocytes has been estimated in vitro by the 3H-thymidine uptake assay. It has been found that porphyrins (endogenous ligands for the mitochondrial benzodiazepine receptor) produce a concentration-dependent inhibition of 3H-thymidine incorporation into the DNA of these cells. Metalloporphyrin-hemin has been observed to evoke a weak inhibitory effect, in a high concentration (10(-4)M), whereas biliverdin, a porphyrins degradation product, was inactive in the same experimental conditions. Those findings indicate that endogenous porphyrins, presumably acting through the mitochondrial benzodiazepine receptor, could regulate the proliferation of mouse spleen lymphocytes in vitro.     

The interaction of tumour-localizing porphyrins with collagen, elastin, gelatin, fibrin and fibrinogen

We have already reported in Balb C mouse transplantable mammary carcinoma, that uroporphyrin I and III are superior as tumour localizers when compared to hematoporphyrin derivative and a derivative thereof, photofrin II. This study compares the binding of porphyrins to proteins which may be found in tumour cells or stroma to investigate whether there is a common binding determinant. Coproporphyrin III and deuteroporphyrin IX which are non-tumour localizing porphyrins, were also part of the comparative study. The interaction of these porphyrins with acid soluble collagen and acid insoluble collagen, elastin, and fibrin was evaluated, and the binding of uroporphyrin isomers I and III and deuteroporphyrin IX to gelatin and fibrinogen, was also determined. The results suggest that collagen, especially the acid soluble form, and gelatin preferentially bind the four porphyrins which localize in mammary carcinoma tissue. The well reported observations that malignant epithelial cells, including breast cancer, produce collagen and contain a rate-limiting enzyme in collagen biosynthesis would support the notion that de novo synthesis of this protein may in part govern the tumour uptake and retention of porphyrins. Elastin, fibrinogen and fibrin showed non-discriminant binding to the porphyrins under study.     

The porphyrin-sensitized photooxidation of horseradish apoperoxidase.

Horseradish apoperoxidase (apoHRP) was reconstituted with various porphyrin derivatives, e.g., ferric, cupric, manganese, and zinc protoporphyrin IX, metal-free protoporphyrin IX, hematoporphyrin IX and deuteroporphyrin IX. The visible absorption spectra of these porphyrin-apoHRP complexes were examined. The time required for maximum development of the new Soret peak after reconstitution was used to measure the rate of porphyrin-apoHRP reconstitution. All of the four metal-protoporphyrins reconstituted with apoHRP at the same rate as metal-free protoporphyrin IX, whereas, for the metal-free porphyrins, the rates of reconstitution were in the order of deuteroporphyrin IX > hematoporphyrin IX > protoporphyrin IX. The porphyrins on the reconstituted porphyrin-apoHRP complexes were used as localized photosensitizers for photodynamic studies. No amino acid residues were oxidized on illumination of the ferric, cupric and manganese protoporphyrin IX-apoHRP complexes due to the paramagnetic properties of these metal ions. With diamagnetic zinc ion, two histidine and one methionine residues were oxidized which was the same as in the protoporphyrin IX- and hematoporphyrin IX-apoHRP complexes. However, only one histidine was destroyed on illumination of the deuteroporphyrin IX-apoHRP complex. The results confirmed the resistance of horseradish peroxidase to photodynamic action and suggested the involvement of at least one histidine residue in the heme environment of horseradish peroxidase.     

Determination of porphyrins and biliverdin in bile and excreta of birds by a single liquid chromatography-ultraviolet detection analysis

Methods developed for porphyrin analysis have low recoveries and/or poor precision for the less polar protoporphyrin IX. We describe a simple method of analysis of porphyrins and biliverdin in bile and excreta of birds based on extraction with HCl 3N: acetonitrile and HPLC/UV analyses. Recoveries were good for protoporphyrin IX and other porphyrins (>79%). Applications of this method showed that porphyrins and biliverdin in birds excreta are mainly of biliary-fecal origin rather than urinary origin. Biliverdin and protoporphyrin IX increased proportionately more than the rest of the porphyrins and coproporphyrin III increased more than coproporphyrin I in the bile of Pb-poisoned mallards.     

The effect on photohaemolysis of variation in the structure of the porphyrin photosensitizer.

A comparison of the photosensitizing ability of a variety of porphyrins for photohaemolysis gives the following order of activity: protoporphyrin greater than deuteroporphyrin, mesoporphyrin, haematoporphyrin dimethyl ester much greater than haematoporphyrin diacetate, haematoporphyrin greater than haematoporphyrin monoacetate, coproporphyrin III, haematoporphyrin derivative, coproporphyrin III tetramethyl ester greater than uroporphyrin I, meso-tetra-(N-methyl-4-pyridinium)porphyrin tetratoluene-p-sulphonate, meso-tetra-(p-carboxyphenyl)porphyrin, protoporphyrin dimethyl ester, meso-tetra-(p-hydroxy-sulphonylphenyl)porphyrin tetrasodium salt, uroporphyrin III, deuteroporphyrin-3,8-disulphonic acid and protohaemin. The results for the metal-free porphyrins are rationalized in terms of solubility and partition properties, and a model is proposed for the incorporation of amphipathic porphyrins into the membrane lipid bilayer. Experiments with erythrocytes from patients with erythropoeitic protoporphyria and with normal erythrocytes to which porphyrin was added in a deuterium oxide medium do not lead to an increase in the rate of photohaemolysis. A possible explanation for this somewhat surprising observation is outlined.     

Interactions with glutathione S-transferases of porphyrins used in photodynamic therapy and naturally occurring porphyrins.

Several naturally occurring porphyrins and porphyrins used in photodynamic therapy inhibit glutathione S-transferase isoenzymes either purified from rat liver or lung or in cytosol from normal and from cancerous (Morris 7288C hepatoma) liver. Although differences occur in the type and amount of transferases in normal and cancerous liver and in the liver of rats bearing an extrahepatic tumour, these enzymes are potential binding sites for porphyrins. Porphyrin structure is an important factor in determining the affinity of binding, as shown by the relative inhibitory effectiveness. Of the dicarboxylic porphyrins in the mixture used clinically, OO'-diacetylhaematoporphyrin and monohydroxyethylmonovinyldeuteroporphyrin are more effective inhibitors than haematoporphyrin and protoporphyrin IX. Of the naturally occurring porphyrins the order of effectiveness is protoporphyrin IX (dicarboxylic) greater than coproporphyrin (tetracarboxylic) greater than uroporphyrin (octacarboxylic) and type I greater than type III isomers of both uroporphyrin and coproporphyrin, and the synthetic tetra-meso-phenylporphinetetrasulphonate is a better inhibitor (apparent Ki = 250 nM) than coproporphyrin, which contains a comparable number of negative charges. In addition, iron-porphyrin chelates are more effective inhibitors of the transferases, with 25-fold decrease in Ki value, than the free porphyrins. These results indicate that one means whereby porphyrins accumulate in tissues is the occupation of intracellular binding sites, such as the transferases. Since porphyrins inhibit the activity of these important detoxifying enzymes, there will be metabolic consequences to the cell.     

The chemical structure of pemptoporphyrin

1. Pemptoporphyrin dimethyl ester was further purified on an alumina column and then crystallized from chloroform and methanol with melting point 215-218 degrees . The absorption spectra in the visible and ultraviolet with molar extinction coefficients are presented. 2. The nuclear-magnetic-resonance spectrum of pemptoporphyrin dimethyl ester in deuterochloroform solution is reported, together with those of several other porphyrins. 3. One vinyl and one hydryl group were found at positions 2 and 4 (or 4 and 2) in the molecule. The structure of pemptoporphyrin is 2(4)-hydryl-1,3,5,8-tetramethyl-4(2)-vinyldeuteroporphyrin IX. 4. Reduction of pemptoporphyrin dimethyl ester with resorcinol gave deuteroporphyrin IX dimethyl ester.     

Formylation of porphyrin platinum complexes

The formylation reaction of platinum complexes of beta-unsubstituted porphyrins was studied. The interaction of deuteroporphyrin IX derivatives with the Vilsmeyer reagent led to the selective formylation of their macrocycles in the beta position. The resulting formyl derivatives of the porphyrins are of interest for fluorescent immunoassay.     

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.     

Porphyrin content of the cysticercus of Taenia solium

The strong red fluorescence of the cysticercus of Taenia solium depends on the presence of several porphyrins in the vesicular fluid of the parasite: probably protoporphyrin IX, coproporphyin I or III, and 2 decarboxylated porphyrins intermediate between uroporphyrin and coproporphyrin. Cyst porphyrins associated to form conglomerates of high molecular weight that dissociated in acid solutions and were not antigenic themselves nor associated with antigenic molecules. An appreciable fraction of the porphyrins was capable of undergoing oxidation and reduction, indicating that some of the porphyrins were complexed with metal ions. The metabolic basis for the accumulation of porphyrins is unknown. Preliminary results suggest that conditions deleterious to the cysticercus cause release of porphyrins so that the appearance of porphyrins in the cerebrospinal fluid of neurocysticercotic patients may prove useful in monitoring therapeutic attacks on the parasite.     

Spectrofluorimetric study of porphyrin incorporation into membrane models--evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.     

Simultaneous determination of six urinary porphyrins using liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method without sample pretreatment was developed and validated for determination of porphyrins in samples of canine urine. Acidified urine samples were directly injected into the LC-MS system and a gradient elution program was applied. The mass spectrometer was operated in the multi-reaction monitoring (MRM) mode and six porphyrins were detected with excellent sensitivity and selectivity. The lower limits of quantification were 0.014 nmol/mL for mesoporphyrin IX, coproporphyrin I, 5-carboxylporphyrin, 6-carboxylporphyrin and 7-carboxylporphyrin, and 0.029 nmol/mL for uroporphyrin I. Good ln-quadratic responses of calibration standards over the range 0.01 to 1.0 nmol/mL for mesoporphyrin IX, coproporphyrin I, 5-carboxylporphyrin, 6-carboxylporphyrin and 7-carboxylporphyrin, and 0.02 to 1.0 nmol/mL for uroporphyrin I were demonstrated. This method should be easily adapted through cross-validation for use in determining the effects of chemicals and pharmaceuticals on the urinary excretion profile of porphyrins in preclinical studies with other species, and in assisting the diagnosis of porphyria in clinical studies.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration.

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value deltae (mM-1 - cm-1) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM - min-1 - mg-1 of protein at 37 degrees C, PH 7.4 and optimal substrate concentrations, i.e. 75 muM Fe(III) and 50 muM deuteroporphyrin. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.     

Porphyrin-sensitized photodynamic damage of isolated rat liver mitochondria

The respiration rates and the respiratory control ratios of isolated rat liver mitochondria have been measured following exposure to 0–160 kJ/m² of near-ultraviolet radiation (blacklight) in the presence of low concentrations of porphyrins (0.1–0.2 μmol/l).

Depending on the light dose, the concentration and the type of porphyrin, the following sequence of reactions occurred: uncoupling and inhibition of oxidative phosphorylation, energy dissipation, inhibition of respiration and swelling and disruption of the mitochondria.

The detrimental effects could not be elicited in the absence of oxygen, neither could they be elicited by porphyrins or light alone.

At equimolar concentrations, the effectiveness of the porphyrins as photosensitizers were: deuteroporphyrin > protoporphyrin coproporphyrin > murophorphyrin.

The results may be of importance to explain the skin lesions seen when porphyrins of different hydrophobicity accumulate in the skin.     

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.     

Spectrofluorimetric study of porphyrin incorporation into membrane models - evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK ≈ 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K ≈ 2 · 10⁵ and K ≈ 6 · 10⁶ M⁻¹ (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.