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.     

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.     

Tumour localization of uroporphyrin isomers I and III and their correlation to albumin and serum protein binding

We were the first to report the superiority of uroporphyrin I (UROP I) as a tumour localizer when compared to haematoporphyrin derivative (HPD). In this study, we compared both isomers of UROP, i.e. I and III, in a KHJJ mammary carcinoma mouse model. Six and 18 h after UROP administration, the tumour, skin and gut porphyrin (P) content was quantitated. Tumour UROP I levels were always at least 50% higher than UROP III in tumour, whereas both isomers were barely detectable in the skin and gastrointestinal tract. We then explored the possibility that tumour P uptake might relate in part to the affinity of circulating P to mouse serum proteins (MSP), in particular, the major binding protein constituent, albumin. Copro-P III, deutero-P 2,4 disulphonic acid (DP), proto-P IX (PP) and heptacarboxylic P I (Hepta I) which in our mouse tumour model do not localize in malignant tissue, were compared to UROP I and III. The P was mixed with 0.775 microM human serum albumin (HSA) at different molar ratios (HSA:P range 2-8) and the unbound P concentration quantitated using an Amicon CF-25 membrane cone with centrifugation. The percentage free P was significantly higher for UROP I (92-98%) than III (82-95%) and significantly more than that observed with non-tumour localizing P studied. Similar data were obtained with MSP. This is consistent with the notion that enhanced uptake and retention (particularly UROP I) by malignant neoplastic tissue might reflect a higher affinity for UROP by tumour constituents than by circulating proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.     

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.     

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.     

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution.

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.     

Porphyrin profiles in blood and urine as a biomarker for exposure to various arsenic species.

A sensitive method using HPLC with fluorescence detection has been established for the measurement of porphyrins in biological materials. The assay recoveries were 88.0+/-1.8% for protoporphyrin IX in the blood, and ranged from 98.3+/-2.7% to 111.1+/-7.4% for various porphyrins in the urine. This method was employed to investigate the altered porphyrin profiles in rats after a single dose of various arsenicals including soluble sodium arsenate and sodium arsenite, and the relatively insoluble calcium arsenite, calcium arsenate and arsenic-contaminated soils at dose rates of 5 mg/kg or 0.5 mg/kg body weight. Porphyrin concentrations increased within 2448 hr after the arsenic treatment in blood and urine. Protoporphyrin IX is the predominant porphyrin in the blood. In rats administered 5 mg As(III)/kg body weight, protoporphyrin IX concentration elevated to 123% of the control values in rats, 24 hr after the treatment. Higher increases were recorded in the urinary protoporphyrin IX (253% at 24 hr; 397% on day 2), uroporphyrin (121% at 24 hr; 208% on day 2) and coproporphyrin III (391% at 24 hr; 304% on day 2), while there was no significant increase (109% on day 3) observed in the urinary coproporphyrin I excretion. In rats administered 5 mg As(V)/kg, urinary excretion of protoporphyrin LX, uroporphyrin, coproporphyrin III and coproporphyrin I elevated to the maximum levels by 48 hr with the corresponding percentage values compared to the control being 177%, 158%, 224% and 143%, respectively. In rats dosed with 5 mg As(III)/kg, the increases (expressed as % of the control values) of protoporphyrin IX in the blood were in the order: sodium arsenite (144%) > sodium arsenate (125%) > calcium arsenite (123%) > calcium arsenate. In contrast, there was no significant increase of protoporphyrin IX, when the six arsenic-contaminated cattle dip soils and nine copper chrome arsenate (CCA-contaminated) soils were administered to the rats. Probable explanations are discussed.     

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.     

Interaction of rabbit hemoplexin with copro- and uroporphyrins.

Rabbit hemopexin forms equimolar complexes in vitro with the I and III isomers of both coproporphyrin and uroporphyrin. The apparent dissociation constants (Kd) of these complexes are estimated to be 4-10(-7) M for coproporphyrin-hemopexin and 10(-6) M for uroporphyrin-hemopexin by equilibrium dialysis and quenching of protein fluorescence. Results of competitive binding experiments suggest that all four porphyrins bind at the heme-binding site of hemopexin, and that the relative affinity of rabbit hemopexin for these porphyrins is: deuteroheme greater than coproporphyrin I or III greater than uroporphyrin I or III. These findings provide further evidence that hemopexin may function as a transport protein for circulating coproporphyrins as well as for heme.     

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.     

Induction of ER stress protects gastric cancer cells against apoptosis induced by cisplatin and doxorubicin through activation of p38 MAPK

Porphyromonas gingivalis acquires heme through an outer-membrane heme transporter HmuR and heme-binding hemophore-like lipoprotein HmuY. Here, we compare binding of iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) to HmuY with that of iron(III) protoporphyrin IX (protoheme) and protoporphyrin IX (PPIX) using spectroscopic methods. In contrast to PPIX, mesoheme and deuteroheme enter the HmuY heme cavity and are coordinated by His134 and His166 residues in a fully analogous way to protoheme binding. However, in the case of deuteroheme two forms of HmuY–iron porphyrin complex were observed differing by a 180° rotation of porphyrin about the α-γ-meso-carbon axis. Since the use of porphyrins either as active photosensitizers or in combination with antibiotics may have therapeutic value for controlling bacterial growth in vivo, it is important to compare the binding of heme derivatives to HmuY.     

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.     

Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV

The circulating enzyme, α₂-antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α₂-antiplasmin to a derivative more easily cross-linked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III, and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Amino-terminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.     

Protoporphyrin IX activates the Mg dependent guanylate cyclase from rat liver plasma membranes

In the presence of Mg-GTP, the rat liver guanylate cyclase, in either intact membranes or trypsin solubilized form, was stimulated by protoporphyrin IX 6 to 10-fold. However, when Mn-GTP was the substrate, protoporphyrin IX activated the membrane-bound guanylate cyclase only 50%, in contrast to the marked activation reported for the cytosolic enzyme. Meso- and deuteroporphyrin IX, hematoporphyrin and coproporphyrin III also activated membrane guanylate cyclase while uroporphyrin III, and hemin had no effect. Basal, Mg2+-dependent activity exhibited two classes of catalytic sites with apparent Km values of 2 mM and 0.12 mM. Activation by protoporphyrin resulted in the disappearance of the low affinity sites. The activated enzyme exhibited Michaelis-Menten kinetics and no alteration in its requirement for excess Mg2+. These data indicate that, in the presence of Mg2+, a heme-like structure can interact with the membrane-bound guanylate cyclase and regulate its activity.     

Macrocyclic intermediates in the biosynthesis of porphyrins.

The hepta-, hexa- and penta-carboxylic porphyrins found in the faeces of rats poisoned with hexachlorobenzene have been separated by high-pressure liquid chromatography and characterized largely by spectroscopie methods. Their structures were confirmed by total synthesis, as part of a programme in which eleven of the fourteen hepta-, hexa- and penta-carboxylic porphyrins derived from uroporphyrin III have now been synthesized as their methyl esters. The four isomeric heptacarboxylic and three of the pentacarboxylic porphyrinogens have been incubated with haemolysates of chicken erythrocytes, and they are all converted into protoporphyrin IX but at different rates. On the basis of this and other evidence we conclude that the decarboxylation of uroporphyrinogen III to coproporphyrinogen III is a stepwise process taking place by a preferred pathway (both in normal and abnormal metabolism); the acetic acid groups are decarboxylated in a sequential clockwise fashion starting with that on the D ring and followed by those on the A, B and C rings. In the poisoned rats the uroporphyrinogen decarboxylase enzyme (or group of enzymes) is probably partially inhibited and the pentacarboxylic porphyrinogen with an acetic acid group on ring C accumulates. The latter is then transformed by a side pathway into dehydroisocoproporphyrinogen and thence into dehydroisocoproporphyrin and its congeners.     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

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.     

Properties of zinc uroporphyrin III produced from isopropanol by Arthrobacter hyalinus

In a large amount of porphyrins produced by a bacterium isolated from soil, Arthrobacter hyalinus, cultured in a medium containing isopropanol as the sole carbon source, zinc porphyrins, identified based on the coincidence between their m/z values in LC/MS and the molecular weight of porphyrins, were also found to be produced. Since zinc is easily separated from porphyrins in acid during the esterification of porphyrins, zinc uroporphyrin III was prepared from its octamethyl ester formed by incorporating zinc into the octamethyl ester of uroporphyrin III which was isolated from the culture broth. Its UV spectra, fluorometric spectra, fast atom bombardment (FAB)-mass spectra, and ¹H-NMR and ¹³C-NMR spectra were presented.