Methylene Blue-sensitized Photooxidation Research Articles

ChemInform Abstract: (6E) and (6Z)‐9′‐Aporhodoxanthinone, Novel Carotenoids Produced in Zeaxanthin‐Synthesizing‐Escherichia coli by Redox Stress.

Abstract Recombinant Escherichia coli, carrying the crtE, crtB, crtI, crtY, and crtZ genes from the bacterium Pantoea ananatis in addition to the Haematococcus pluvialis idi gene, can dominantly produce zeaxanthin. Vigorous rotary-shaking culture of the recombinant cells to promote strong aeration led to the production of two structurally similar polar carotenoids [1 (3.7%) and 2 (2.9%)] and eschscholtzxanthone (2.3%), along with zeaxanthin (72.6%), β-cryptoxanthin (7.3%), β-carotene (7.1%), and rubixanthin (2.1%). Carotenoids 1 and 2 were purified, and their structures were determined as (6E)-9′-apo-rohodoxanthinone and (6Z)-9′-apo-rhodoxanthinone, respectively, based on UV–vis, MS, 1H NMR, and 13C NMR spectral data. These novel compounds were shown to exhibit excellent singlet oxygen-quenching activity by the methylene blue-sensitized photooxidation system.

(6E) and (6Z)-9′-Aporhodoxanthinone, novel carotenoids produced in zeaxanthin-synthesizing-Escherichia coli by redox stress

Recombinant Escherichia coli, carrying the crtE, crtB, crtI, crtY, and crtZ genes from the bacterium Pantoea ananatis in addition to the Haematococcus pluvialis idi gene, can dominantly produce zeaxanthin. Vigorous rotary-shaking culture of the recombinant cells to promote strong aeration led to the production of two structurally similar polar carotenoids [1 (3.7%) and 2 (2.9%)] and eschscholtzxanthone (2.3%), along with zeaxanthin (72.6%), β-cryptoxanthin (7.3%), β-carotene (7.1%), and rubixanthin (2.1%). Carotenoids 1 and 2 were purified, and their structures were determined as (6E)-9′-apo-rohodoxanthinone and (6Z)-9′-apo-rhodoxanthinone, respectively, based on UV–vis, MS, 1H NMR, and 13C NMR spectral data. These novel compounds were shown to exhibit excellent singlet oxygen-quenching activity by the methylene blue-sensitized photooxidation system.

Antiphotooxidative activities of elm (Ulmus davidiana Planch) root bark extracts on methylene blue-sensitized photooxidation of lipids and isolation and identification of active compounds

Methanol extracts of elm (Ulmus davidiana Planch) root bark were fractionated and purified to obtain active antiphotooxidative compounds against methylene blue sensitized photooxidation of oil. Crude extracts of elm root bark exhibited a strong protective activity, comparable to α-tocopherol, against methylene blue sensitized photooxidation of oil. Butanol and ethylacetate fractions of crude extracts showed strong antiphotooxidative activities. Four isolated pure compounds were obtained and positively identified as catechin-7-O-d-xylopyranoside (CXP), catechin-7-O-d-apiofuranosid (CAF), catechin (C), and mansonone E. CXP, CAF, and C were identified as active compounds showing strong protective activities against methylene blue sensitized photooxidation. However, mansonone E at a 300 ppm concentration induced a 20% increase in hydroperoxide formation. These 3 compounds are major active compounds for the antiphotooxidative activities of elm root bark extracts against methylene blue sensitized photooxidation of oils.

Methanolic extract of Coptis japonica Makino reduces photosensitized oxidation of oils

Antioxidative activities of methanolic extracts of 47 plants on the methylene blue-sensitized or chlorophyll-sensitized photooxidations of linoleic acid were studied to select the plant species which possess the strong antioxidative activity in sensitized photooxidation of oil. The methanol extract of Coptis japonica Makino showed the strongest antioxidative activity in both methylene blue-sensitized and chlorophyll-sensitized photooxidations of linoleic acid. The study on the extracting solvent effects indicated that the antiphotooxidative components in the Coptis japonica Makino possess strong polar property, and are easily extracted with highly polar solvent (methanol). The methanol extract of Coptis japonica Makino was fractionated into three fractions (ethyl ether fraction, ethyl acetate fraction and butanol fraction) by liquid–liquid partitioning fractionation. Among the tested fractions, the butanol fraction showed strongest antioxidative activity in both chlorophyll-sensitized and methylene blue-sensitized photooxidation of linoleic acid. The butanol fraction also significantly inhibited the photooxidation of model food emulsion (50% soybean oil emulsion). The treatment with 0.3% (w/w) butanol fraction resulted in 64.9% inhibition of photooxidation of the model food emulsion during 60 h fluorescent light illumination.

Singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis

The singlet oxygen quenching activities of carotenoids, β-carotene, free astaxanthin, its monoester and its diester, were examined in vitro by a simple and rapid method for the measurement of Methylene Blue-sensitized photooxidation of linoleic acid in the hexane/ethanol solvent system. The concentrations of carotenoids, β-carotene, free astaxanthin, its mono- and di-ester, required for 50% inhibition of lipid oxidation were 40, 8, 9, and 0 μM in 100% ethanol and, 14, 16, 10, and 7 μM in 50% (v/v) hexane in ethanol, respectively. Astaxanthin esters function as powerful antioxidant agents under both hydrophobic and hydrophilic conditions.

Determination of singlet oxygen-specific versus radical-mediated lipid peroxidation in photosensitized oxidation of lipid bilayers: effect of beta-carotene and alpha-tocopherol.

Photosensitized oxidation reactions damage tissue by catalyzing the formation of oxyradicals and singlet oxygen. beta-Carotene is hypothesized to exert photoprotective effects by quenching singlet oxygen formed by Type II reactions and by scavenging free radicals formed by Type I reactions. beta-Carotene antioxidant mechanisms were studied in a phospholipid membrane model of photooxidation with a new isotope dilution gas chromatography-mass spectrometry (GC-MS) assay that quantitatively distinguishes singlet oxygen-mediated and radical-mediated lipid peroxidation. This assay measures 9- and 10-hydroxylinoleate methyl esters and was used to generate photooxidation profiles for the photosensitizers methylene blue, Rose Bengal, and tetraphenylporphine. These profiles indicate a shift from Type II to Type I photooxidation mechanisms in later stages of photooxidation. beta-Carotene (0.45 mol %) inhibited singlet oxygen-mediated lipid peroxidation at early stages of methylene blue-sensitized photooxidation. Production of radical-mediated products increased faster than singlet oxygen-mediated products at later stages. beta-Carotene-5,8-endoperoxide, a specific marker for singlet oxygen oxidation of beta-carotene in solution, was unstable under the incubation conditions and was not detected in this system. alpha-Tocopherol (0.45 mol %) was ineffective in inhibiting photosensitized lipid peroxidation, whereas 4.5 mol % alpha-tocopherol inhibited almost all radical-mediated lipid peroxidation as well as early-stage singlet oxygen-mediated lipid peroxidation. Cumene hydroperoxide stimulated radical-mediated lipid peroxidation, indicating that accumulation of hydroperoxides from Type II photooxidation may enhance Type I reactions. These data suggest that singlet oxygen quenching, rather than radical scavenging reactions, accounts for the photoprotective actions of beta-carotene.

Riboflavin-sensitized photooxidation of ascorbic acid: kinetics and amino acid effects

The study on the effects of riboflavin contents and light intensity on the riboflavin-sensitized decomposition of ascorbic acid indicated that light and riboflavin accelerated the decomposition of ascorbic acid. The relative photosensitizing activity of riboflavin, methylene blue and protoporphyrin IX was 21:15:1 at 1–2 ppm. The rate constants for reaction of ascorbic acid with singlet oxygen at pH 7·5, 6·0 and 4·5 were 6·63 × 10 8, 5·77 × 10 8 and 5·27 × 10 8 M −1 s −1, respectively. Cysteine showed strong antioxidant activity on both riboflavin- and methylene blue-sensitized photooxidation of ascorbic acid and its antioxidant effect was concentration-dependent. Alanine and phenylalanine at 0·1% concentration showed antioxidant effects on the riboflavin-sensitized photooxidation of ascorbic acid but prooxidant effects on the methylene blue-sensitized photooxidation. Tyrosine (0·1%) exhibited strong prooxidant activity on both the riboflavin- and methylene blue-sensitized photooxidation of ascorbic acid. However, tryptophan (0·1%,) showed antioxidant or prooxidant activity on the photooxidation of ascorbic acid depending on the storage time.

The Effecxt of Methylene Blue-Sensitized Photodynamic Treatment on Bladder Cancer Cells: A Further Study on Flow Cytometric Basis

The cytotoxic effect mediated by methylene blue-sensitized photoinactivation on bladder cancer cells has been well known for years, but the actual mechanism is still not clear. Specific fluorescence stains were used in a multiparameter approach. The changes in cellular size, granularity, protein content, RNA and DNA were analyzed by flow cytometry. The results showed that, in addition to cell size, all the other parameters, including cellular granularity, protein, RNA and DNA, diminished following photodynamic therapy. The cytotoxic efficacy is closely related to time of stain and illumination. The DNA and cell cycle were affected simultaneously with decreased DNA content and arrest in the G0/G1 phase. These multistage processes occurred synchronously after the phototherapy. Our results suggest that the methylene blue-sensitized photooxidation of bladder cancer cells is carried out by multimedia reactions through which the intracellular proteins (enzymes in mitochondria), RNA (ribosomes) and DNA (chromatin) decrease with loss of cytoplasmic granularity.

ChemInform Abstract: The Methylene Blue‐Sensitized Photooxidation of Quadricyclane, Reinvestigation.

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The Methylene Blue-Sensitized Photooxidation of Quadricyclane, Reinvestigated

Abstract Photooxygenation of quadricyclane(1) was shown to proceed via the electron transfer mechanism on the basis of the structure revision of a methoxy alcohol. Photoisomerization of 1 to norbornadiene occurred during the irradiation.

Autoxidation and photosensitized oxidation of methyl eicosapentaenoate: Secondary oxidation products.

Methyl eicosapentaenoate (methyl 5, 8, 11, 14, 17-eicosapentaenoate) was subjected to autoxidation and methylene blue-sensitized photooxidation. The secondary oxidation products were separated, and characterized by gas chromatography-mass spectrometry. The autoxidation products included hydroperoxy endoperoxide isomers, prostaglandin-like hydroperoxy bicyclic endoperoxide isomers and 5, 18-dihydroperoxide. The photosensitized oxidation products included only dihydroperoxide isomers as the secondary products.

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase ( M r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of L - alanyl-p- nitroanilide exhibited a bell-shaped pH dependence for log V max/ K m(p K 1 = 6.35; p K 2 = 8.50) while the log V max versus pH profile showed only an acid limb (p K = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.

Photo-oxidation and photoprotection in the IXγ bile pigment series: A comparison of the photoprotective roles of pterobilin, phorcabilin and sarpedobilin in vitro

Pterobilin ( L9, biliverdin IXγ), a blue green bile pigment from butterflies, is an effective photoprotector of the methylene blue-sensitized photo-oxidation of bilirubin (both as dimethyl esters). The observed effect is of the same order as that observed for the isomerie biliverdins IXα, IXβ, and IXδ ( L4, L8, and L10, respectively). However the dimethyl esters of phorcabilin ( 1) and sarpedobilin ( 2), the cyclized derivatives of pterobilin, are unable to provide photoprotection under the same conditions. The possible significance of this result is considered.

Inactivation of d-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation

Cell-free d-glucosyltransferase of d-glucose-grown Streptococcus mutans AHT was completely inactivated in the presence of 0.002% of Methylene Blue at 25° and pH 7.0 after illumination with a 150-W incandescent lamp. The rate of inactivation was decreased at pH values <7.0. Histidine was the only amino acid residue modified to a significant extent, and the rates of oxidation of histidine residues and loss of enzyme activity closely agreed. Production of both water-insoluble and -soluble d-glucan fractions from sucrose by the oxidized d-glucosyltransferase preparations was significantly inhibited. Photooxidation with 0.002% of Rose Bengal at pH 7.0 or higher also induced complete inactivation of the d-glucosyltransferase. These results strongly suggest that the imidazole portion of histidine may function as part of the active sites of both d-glucosyltransferase isozymes of S. mutans AHT, which are responsible for the synthesis of (1→3)- and (1→6)-α- d-glucosidic linkages. The d-glucosyltransferases from S. mutans 6715 and AHT-mutant Ml, and Streptococcus sanguis ATCC 10558 were also almost completely inactivated by Methylene Blue-sensitized photooxidation.

Temperature- and substituent-dependence in the photosensitized oxygenation of N,N-disubstituted hydrazones

Methylene blue-sensitized photooxidation of N,N-disubstituted hydrazones 1a-f gave various products 2-9 , depending on the reaction temperature and N-substituents. The major reaction modes from the photo-oxidation of N,N-diphenylhydrazones were α-oxidation at room temperature and C=N cleavage at −78 °C in contrast to C=N cleavage as an only reaction mode for the photooxidation of N,N-dimethylhydrazones at either room temperature or −78 °C.

Electrophorus acetylcholinesterase. Biochemical and electron microscope characterization of low ionic strength aggregates.

The "tailed" molecules of Electrophorus (electric eel) acetylcholinesterase aggregate under conditions of low ionic strength. These aggregates have been studied by sedimentation analysis and high-resolution electron microscopy. They consist of bundles of at least half a dozen molecules, the tails of which are packed side by side, to form the core of the structure. Although aggregation is normally fully reversible, aggregates were irreversibly stabilized by methylene blue-sensitized photo-oxidation. This process was shown to consist of a singlet oxygen oxidation reaction and probably involves methionine or histidine residues. It did not modify the structural or hydrodynamic characteristics of the aggregates.

Covalent cross-linking of ribosomal RNA and proteins by methylene blue-sensitized photooxidation

Ribosomal proteins are covalently cross-linked to ribosomal RNA by irradiation with visible light in the presence of methylene blue and O 2. Proteins S3, S4, S5 and S7 from the 30 S subunit of Escherichia coli ribosomes and L2 and L3 from the 50 S subunit are among the cross-linked proteins. S3 and S5 had not previously been identified as RNA-binding proteins.

Superoxide dismutase from an anaerobic photosynthetic bacterium Chromatium vinosum

Superoxide dismutase of anaerobic purple sulfur bacterium, Chromatium vinosum, was purified to a homogeneous state. The enzyme contains two atoms of iron per mole and has a molecular weight of 41,000. It is composed of two identical subunits. Amino acid composition, absorption spectra, and the reaction rate constant with O 2 − are also similar to those of the Fe-superoxide dismutases from aerobes. The enzyme is sensitive to hydrogen peroxide and methylene blue-sensitized photooxidation. The functional and evolutional aspects of superoxide dismutase in anaerobes are discussed.

Cleavage of tryptophanyl peptide bonds in cytochrome b5 by cyanogen bromide.

Quantitative cleavage of peptide bonds adjacent to tryptophanyl and methionyl residues in the polar moiety of cytochrome b5 was effected using cyanogen bromide in the presence of heptafluorobutyric and formic acids. Application of this method to native cytochrome b5 resulted in cleavage at tryptophanyl and methionyl residues in the polar and membranous segments in high yield. Amino acid analysis of peptides isolated from such digests indicated that tyrosine was modified and the derivative eluted in a position preceding lysine; however, the color constant with ninhydrin remained unchanged. Hydriodic acid hydrolysis of the phenylthiohydantoin derivative of the modified tyrosine regenerated the parent amino acid. Peptides containing the altered tyrosine remained susceptible to chymotryptic cleavage at this residue. Cleavage of methionyl bonds could be prevented by methylene blue-sensitized photooxidation prior to cyanogen bromide/anhydrous heptafluorobutyric acid treatment. The conditions employed for photooxidation were selective for methionyl residues, and the yield and specificity of tryptophanyl cleavage by the cyanogen bromide/anhydrous heptafluorobutyric acid method was unaffected.

Studies on the active site of papain. VII. States of tryptophan residues.

1) The present research has been planed to clarify the states of tryptophan residues in papain by means of N-bromosuccinimide (NBS) oxidation and photooxidation. 2) Only a tryptophan residue was exclusively modified by NBS oxidation with the loss of enzyme activity. 3) This residue was not affected by methylene blue-sensitized photooxidation. 4) Two of five tryptophan residues were not affected by NBS oxidation and photooxidation. 5) The first NBS-oxidizable tryptophan residue is considered to exist in or nearby the active site of papain. 6) The second and the third NBS-oxidizable tryptophan residues are considered to be identical with the photooxidizable residues. 7) The state of tryptophan residues of papain was illustrated schematically.