A search for porphyrin biomarkers in nonesuch shale and extraterrestrial samples

An organic solvent extract of billion year old Nonesuch Shale was examined for porphyrins by means of fluorometry and high resolution mass spectrometry. It appears to contain at least three or more classes of porphyrins, one similar to tetraphenyl porphin and the others more complex. Many are apparently chelated with copper, nickel, zinc, iron and vanadyl and are highly aromatic. We have also examined the extracts of Apollo 11, 12 and 14 surface fines for pophyrins by spectrophotofluorometry but we found none even though our method was capable of detecting 10⁻¹³ moles per gm of sample. Lunar Science Institute Contribution.     

Research for amino acids in lunar samples

Water extracts of lunar fines were analyzed for amino acids by a gas-liquid chromatographic technique whereby amino acids were converted to the N-trifluoroacetyln-butyl, esters prior to analysis. The lunar material studied included both Apollo 14 (14240 SESC and 14298) and Apollo 12 (12023) samples. The water extract of the special Apollo 14 sample (14240 SESC) was analyzed both for free and bound amino acids (hydrolysis with 6 N hydrochloric acid). In both the hydrolyzed and unhydrolyzed extracts, the amino acids were not observed above background levels.The analysis of Apollo 12 and 14 samples (12023 14298) yielded similar results. Detection limits were established at 300 pg to 1 ng for different amino acids. A large chromatographic peak with a retention temperature of 126°C was observed on analysis of sample, (12023); it was identified as oxalic acid by GC-MS. The concentration of amino acids in the Apollo 14 SESC samples processed and analyzed in the joint experiments at Ames by GLC and IEC were found to be extremely low (glycine at 3 to 4 ng g^–1). As the quantities were so minute, these identifications could not be confirmed by GLC-MS and therefore should still be considered as tentative. Other studies included the analysis of performance standards at the 2 to 6 ng level of each of 17 amino acids, and the analysis of 5 ml of H₂O containing 2 ppb of each amino acid. Recovery of amino acids added to lunar fines were conducted at the 10, 50, and 70 ng level of each amino acid with 50 to 70 mg of lunar material. The recoveries varied from as high as 80% for some of the aliphatics to complete loss of the amino acids ornithine and lysine.Contributed from Missouri Agricultural Experiment Station Journal Series No. 6255. Approved by the Director. Supported in part by grants from the National Aeronautics and Space Administration (NGR 26-004-011) and the Experiment Station Chemical Laboratories.     

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.     

Manganese substituted Compound I of cytochrome P450 biomimetics: A comparative reactivity study of Mn-oxo versus Mn-oxo species

Manganese-oxo porphyrins have been well studied as biomimetic models of cytochromes P450 and are known to be able to catalyze substrate hydroxylation reactions. Recent experimental studies [J.Y. Lee, Y.-M. Lee, H. Kotani, W. Nam, S. Fukuzumi, Chem. Commun. (2009) 704] showed that Mn(V)-oxo porphyrins react rapidly with 10-methyl-9,10-dihydroacridine (AcrH₂) via a proton-coupled-electron-transfer followed by an electron transfer. In this work, we present a computational study on the reactivity patterns of Mn(V)-oxo and Mn(IV)-oxo with respect to AcrH₂. This study shows that although both oxidants are capable of hydroxylating AcrH₂, the Mn^V-oxo species is the more active oxidant. We have generalized these observations with thermodynamic cycles that explain the reaction mechanisms and electron transfer processes. For the Mn^V-oxo mechanism the reactions proceed with a fast spin state crossing from the ground state singlet to the triplet spin state prior to a hydrogen atom transfer followed by another electron transfer. The present results are fully consistent with previous studies on iron-oxo porphyrins and manganese-oxo porphyrins and shows that the interplay of low lying singlet and triplet spin state surfaces influences the reaction mechanisms and kinetics.     

Mg-protoporphyrin-IX and delta-aminolevulinic acid synthesis from glutamate in isolated greening chloroplasts. Mg-protoporphyrin-IX synthesis

A crude plastid preparation from greening cucumber cotyledons was able to accumulate Mg-protoporphyrin-IX when incubated in the presence of glutamate. ¹⁴C from l-[U-¹⁴C]glutamate was incorporated into the porphyrin. The product was identified by its emission and excitation fluorescence spectra and by its chromatographic behavior on cellulose thin layers. The biosynthesis had a marked requirement for ATP and O₂. α,α′-Dipyridyl, a metal ion-chelating agent which had been shown to stimulate the synthesis of Mg porphyrins and phorbins in etiolated bean leaves and other whole tissues, stimulated likewise the in vitro synthesis of Mg-protoporphyrin-IX by isolated greening chloroplasts. Freezing and thawing, which destroy organelle integrity, abolished the ability of these isolated plastids to biosynthesize Mg-protoporphyrin-IX.     

A rapid,simple method for obtaining radiochemically pure hepatic heme

Radioactivity-labelled heme has usually been isolated from liver to which unlabelled carrier has been added by long, laborious techniques involving organis solvent extraction followed by crystallization. We have devised a simpler, rapid method for obtaining radiochemically-pure heme synthesized in vivo in rat liver from δ-amino[4-¹⁴C]levulinate, by modifying our previous procedure (Bonkowsky et al. (1975) J. Clin. Invest. 56, 1139–1148). This method, in which the heme is extracted into ethyl acetate/glacial acetic acid and in which porphyrins are removed from the heme-containing organic phase with HCl washes, does not require addition of carrier heme. The new method gives better heme recoveries than and heme specific activities identical to, those obtained using the crystallization method. In this new method heme must by synthesized from δ-amino[4-¹⁴C]levulinate; it not satisfactory to use [2-¹⁴C]-glycine substrate because non-heme counts are isolated in the heme fraction.     

Coordination of bismuth and lead in porphyrins: Towards an in-situ generator for α-radiotherapy?

In order to investigate the possible coordination of both bismuth(III) and lead(II) by porphyrins for an application in alpha-radioimmunotherapy, several series of functionalized bismuth porphyrins have been synthesized and their stabilities in acidic medium were compared.Where unfunctionalized porphyrins are not suitable for such a purpose, strapped hanging carboxylate porphyrins are stable and allow a rapid coordination of both bismuth and lead. This property could lead to the preparation of an in-situ generator of alpha particles by a pre-complexation of ²¹²lead, the radioactive parent of ²¹²bismuth, in the ligand.     

Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log P_OW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.     

Catabolism of porphobilinogen by etiolated barley leaves

When [2,4-¹⁴C]porphobilinogen (PBG) or [2 (aminomethyl),5-¹⁴C]PBG is administered to etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness, label becomes incorporated into CO₂, organic and amino acids, sugars, lipids, and proteins during a 4-hour incubation. Less than 1% of the label, however, is incorporated into porphyrins. The rate of ¹⁴CO₂ evolution from leaves fed [2,4-¹⁴C]PBG is strongly inhibited by anaerobiosis but is unaffected by aminooxyacetic acid, while the rate of ¹⁴CO₂ evolution from [2(aminomethyl),5-¹⁴C]PBG is strongly inhibited by aminooxyacetic acid but is not affected by anaerobiosis.     

86 Characterization and differentiation of binding modes of water-soluble porphyrins at complexation with DNA

The influence of water-soluble cationic meso-tetra-(4?N-oxyethylpyridyl)porphyrin (H₂TOEPyP4) and it’s metallocomplexes with Ni, Cu, Co, and Zn on hydrodynamic and spectral behavior of DNA solutions has been studied by UV/Vis absorption and viscosity measurement. It was shown that the presence of planar porphyrins such as H₂TOEPyP4, NiTOEPyP4, and СuTOEPyP4 leads to an increase in viscosity at relatively small concentrations, and then decrease to stable values. Such behavior is explained by intercalation of these porphyrins in DNA structure because the intercalation mode involves the insertion of a planar molecule between DNA base pairs which results in a decrease in the DNA helical twist and lengthening of the DNA. Further decrease of viscosity is explained by the saturation intercalation sites and occurs outside the binding mode. But, in the case of porphyrins with axial ligands such as CoTOEPyP4 and ZnTOEPyP4, the hydrodynamic parameters decrease, which is explained by self-stacking of these porphyrins in DNA surface. This data are proved by spectral measurements. The results obtained from titration experiments were used for calculation of binding parameters: the binding constant K _b and the number of binding sites per base pair n. Obtained data reveal that K _b varies between 3.4 and 5.4?×?10⁶?M^?1 for a planar porphyrins, a range typical for intercalation mode interactions, and 5.6?×?10⁵?M^?1 and 1.8?×?10⁶?M^?1 for axial porphyrins. In addition, the exclusion parameter n also testifies that at intercalation, (n～2) the adjacent base pairs are removed to place the planar molecules, and for outside binders to pack on the surface needs too few places (n～0.5–1). It is apparent that the binding is somewhat stronger at intercalation. The viscometric and spectrophotometric measurements are in good agreement.     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

A series of meso-tris(N-methyl-pyridiniumyl)-(4-alkylamidophenyl) porphyrins: Synthesis,interaction with DNA and antibacterial activity

A series of meso-5,10,15-tris(N-methyl-4-pyridiniumyl)-20-(4-alkylamidophenyl) porphyrins were synthesized by derivatizing the amino group on the phenyl ring with the following hydrophobic groups: –C(O)C₇F₁₅, –C(O)CHCH₂, C(O)CH₃, –C(O)C₇H₁₅, and –C(O)C₁₅H₃₁. The cationic tris-pyridiumyl porphyrin core serves as a DNA binding motif and a photosensitizer to photomodify DNA molecules. The changes of the UV–Vis absorption spectra during the titration of these porphyrins with calf thymus DNA revealed a large bathochromic shift (up to 14 nm) and a hypochromicity (up to 55%) of the porphyrins Soret bands, usually considered as proof of porphyrin intercalation into DNA. Association constants (K) calculated according to the McGhee and von Hippel model, were in the range of 10⁶–10⁷ M⁻¹. An increase in hydrophobicity of the substituents at the 20−meso-position produced higher binding affinity. These porphyrins caused photomodification of the supercoiled plasmid DNA when a green laser beam at 532 nm was applied. Those with higher surface activity acted more efficiently as DNA photomodifiers. The porphyrin with a perfluorinated alkyl chain (–COC₇F₁₅) at the meso-20-position inhibited the growth of gram-positive bacteria (S. aureus, or S. epidermidis). Other porphyrins exhibited moderate activity against both gram-negative and gram-positive organisms.     

Boronated derivatives of chlorin <Emphasis Type="Italic">e</Emphasis><Subscript>6</Subscript> and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e ₆ and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e ₆ by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec⁻¹, respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.     

A quest for porphyrins in lunar soil: Samples from Apollo 11, 12 and 14

Pigments exhibiting porphyrin-like behavior were detected in samples from Apollo 11 and 12. Those from Apollo 11 appeared to be directly related to the rocket exhaust of the descent engine; those from Apollo 12 from indigenous lunar material. Porphyrin-like pigments were not detected in a sample from Apollo 14.     

In vitro effect of lead acetate on human erythropoietic progenitors

Lead is known to induce hematological disturbances resulting from abnormalities in cell differentiation and hemoglobin synthesis during hematopoiesis. The aim of the present work was to study human erythropoiesisin vitro in the presence of lead. Human erythroblastic progenitors, burst-forming units–erythroid (BFU-E), were exposed to lead acetate at increasing concentrations during 14 days of culture. Hematotoxicity was evaluatedin vitro according to proliferation and differentiation of cell colonies arising from BFU-E development. The ability of cells to synthesize proteins, porphyrins, and hemoglobin was measured by spectrophotometric tests and by high-pressure liquid chromatography (HPLC). Results showed that in the presence of 10^–3 mol/L lead acetate, no hemoglobinized cells were observed in culture and no fluorescent porphyrins were detected in cells. Up to 10^–3 mol/L, lead acetate is not cytotoxic, i.e., it does not induce cell destruction. The present work demonstrates that lead acetate interferes with the porphyrin synthesis of human erythroblastic progenitors in vitro. The decrease of porphyrin content with 10^–5 mol/L lead acetate suggest that δ-aminolevulinic acid dehydratase can be inhibited by lead acetate duringin vitro erythropoiesis. In vivo erythropoiesis occurs in the bone marrow. As about 95% of the body burden of lead in adults is located in the bones with a biological half-life of some years, the concentration of lead acetate found to block porphyrin synthesis in vitro has to be compared with in situ bone marrow lead concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Haem synthase and cobalt porphyrin synthase in various micro-organisms

1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co²⁺ ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co²⁺ ions into these two porphyrins is rapid. These extracts are unable to insert Mn²⁺, Zn²⁺, Mg²⁺ or Cu²⁺ ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co²⁺ and Fe²⁺ ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co²⁺ ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.     

Synthesis, characterization and electrode adsorption studies of porphyrins coordinated to ruthenium(II) polypyridyl complexes

Two new porphyrins, meso-tris-3,4-dimethoxyphenyl-mono-(4-pyridyl)porphyrin (H₂MPy3,4DMPP) and meso-tris-3-methoxy-4-hydroxyphenyl-mono-(4-pyridyl)porphyrin (H₂MPy3M4HPP), and their ruthenium analogs obtained by coordination of [Ru(bpy)₂Cl]⁺ groups (where bpy = 2,2′-bipyridine) to the pyridyl nitrogens have been synthesized and studied by electronic absorption spectroscopy, cyclic voltammetry and spectroelectrochemistry. These ruthenated porphyrins couple Ru chromophores to porphyrins containing electroactive meso-substituents. The highest energy electronic absorption for the ruthenated complexes is assigned as a bpy(π) → bpy(π*) intraligand charge transfer while the next lowest energy electronic absorption is assigned as Ru(dπ) → bpy(π*) metal-to-ligand charge transfer (MLCT) transition. The Ru^III/II couples occur at approximately 0.95 V versus the SHE reference electrode in acetonitrile solutions. The first oxidation of the porphyrin is localized on the 3,4-dimethoxyphenyl and 3-methoxy-4-hydroxyphenyl substituents, respectively. Electroactive surfaces result from adsorption of these compounds onto glassy carbon electrodes followed by anodic cycling in acidic media.     

Porphyrin Biosynthesis in Cell-free Homogenates from Higher Plants

The porphyrin and phorbin biosynthetic activity of etiolated cucumber (Cucumis sativus, L.) cotyledons was compared to that of cotyledonary homogenates. Etiolated cotyledons incubated with δ-aminolevulinic acid accumulate protoporphyrin, coproporphyrin, small amounts of Mg protoporphyrin monoester, and trace amounts of uroporphyrin. They also incorporate 4-¹⁴C-δ-aminolevulinic acid into free porphyrins, protochlorophyllide, protochlorophyllide phytyl ester, and Mg protoporphyrin monoester. Homogenates incubated with δ-aminolevulinic acid likewise accumulate coproporphyrin, uroporphyrin, Mg coproporphyrin, and trace amounts of protoporphyrin. They also incorporate 4-¹⁴C-δ-aminolevulinic acid into Mg protoporphyrin monoester, Mg coproporphyrin, and free porphyrins. However, the capacity to synthesize protochlorophyllide and protochlorophyllide phytyl ester is lost and the endogenous protochlorophylls gradually disappear. Mg protoporphyrin monoester represents the terminal biosynthetic step in this cell-free system.     

SNMIB/Apollo protects leading‐strand telomeres against NHEJ‐mediated repair

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell''s inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo''s conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo^−/−ATM^−/− MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5′–3′ exonuclease function of SNM1B/Apollo in the generation of 3′ single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.     

Quantitative and Qualitative Microbiological Profiles of the Apollo 10 and 11 Spacecraft

Microbiological profiles were determined for surfaces of the command module, lunar module (ascent and descent stages), instrument unit, Saturn S-4B stage, and the spacecraft lunar module adapter of the Apollo 10 and 11 spacecraft. Average levels of contamination of the command module were 2.1 x 10(4) and 2.7 x 10(4) microorganisms per ft(2) for Apollo 10 and 11, respectively. With the exception of the exterior surfaces of the ascent stage of the lunar module and the interior surfaces of the command module, average levels of microbial contamination on all components of the Apollo 11 were found to be lower than those observed on Apollo 10. For each Apollo mission, approximately 2,000 colonies were picked from a variety of media and identified. The results showed that approximately 95% of all isolates were those considered indigenous to humans; the remaining were associated with soil and dust in the environment. However, the ratio of these two general groups varied depending on the degrees of personnel density and environmental control associated with each module.