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Andrés H. Thomas - One of the best experts on this subject based on the ideXlab platform.
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Solar radiation exposure of Dihydrobiopterin and biopterin in aqueous solution
Solar Energy, 2014Co-Authors: Adriana Ipiña, Carolina Castaño, M. Laura Dántola, Andrés H. ThomasAbstract:Abstract Dihydrobiopterin (H2Bip) and biopterin (Bip) accumulate in the skin of patients suffering from vitiligo, whose lack of melanin causes a deficient protection against UV radiation. H2Bip and Bip undergo photooxidation upon UV irradiation and the former acts as a photosensitizer of biomolecules. The aim of this work is to study the photochemical reactions of these compounds triggered by sunlight under outdoor conditions. H2Bip and Bip in aqueous solutions were exposed to sunlight in La Plata city (34.90°S, 57.92°W) from Spring equinox to near Summer solstice and the spectral solar irradiance was recorded with a high-resolution spectrometer. The photochemical reactions were followed by HPLC and UV-Vis spectrophotometry. Upon sun exposure, excitation of H2Bip leads to the formation of dimers and to its oxidation to Bip, which, in turn, is photooxidized into 6-formylpterin (Fop). Further excitation induces the oxidation of Fop to 6-carboxypterin, which is much more photostable than Bip and Fop and then it is accumulated in the solution. Rates of reactant consumption were determined under different weather conditions and the corresponding quantum yields were also calculated. We have demonstrated that solar radiation causes significant oxidation of the pterin derivatives investigated within a few minutes, even in cloudy days. Finally, the biological implications of our results are discussed.
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Characterization and reactivity of photodimers of dihydroneopterin and Dihydrobiopterin.
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2012Co-Authors: Mariana Vignoni, Franco M. Cabrerizo, Carolina Lorente, Esther Oliveros, Rosa Erra-balsells, Andrés H. ThomasAbstract:7,8-Dihydrobiopterin (H2Bip) and 7,8-dihydroneopterin (H2Nep) belong to a class of heterocyclic compounds present in a wide range of living systems. H2Bip accumulates in the skin of patients suffering from vitiligo, whereas H2Nep is secreted by human macrophages when the cellular immune system is activated. We have investigated the photochemical reactivity of both compounds upon UV-A irradiation (320–400 nm), the chemical structures of the products and their thermal stability. The study was performed in neutral aqueous solutions. The reactions were followed by UV/Visible spectrophotometry and HPLC and the products were analyzed by means of electrospray ionization mass spectrometry and 1H-NMR. Excitation of H2Bip and H2Nep leads to the formation, in each case, of two main isomeric dimers. The latter compounds undergo a thermal process that may consist in a retro [2 + 2]-cycloaddition and hydrolysis to yield the reactant (H2Bip or H2Nep) and a product that has incorporated a molecule of H2O.
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Photochemistry of Dihydrobiopterin in aqueous solution
Organic & biomolecular chemistry, 2009Co-Authors: Mariana Vignoni, Franco M. Cabrerizo, Carolina Lorente, Catherine Claparols, Esther Oliveros, Andrés H. ThomasAbstract:Dihydrobiopterin (H2Bip) and its oxidized analogue, biopterin (Bip), accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder in which the protection against UV radiation fails. The photochemistry of H2Bip was studied in neutral aqueous solutions upon UV-A irradiation (320–400 nm) at room temperature. The photochemical reactions were followed by UV/vis spectrophotometry, HPLC and enzymatic methods for hydrogen peroxide (H2O2) determination. Photoproducts were analyzed by means of electrospray ionization mass spectrometry. Under anaerobic conditions, excitation of H2Bip leads to the formation of at least two isomeric dimers with molecular masses equal to exactly twice the molecular mass of the reactant. This reaction takes place from the singlet excited state of the reactant. To the best of our knowledge, this is the first time that the photodimerization of a dihydropterin is reported. In the presence of air, the dimers are again the main photoproducts at the beginning of the reaction, but a small proportion of the reactant is converted into Bip. As the reaction proceeds and enough Bip accumulates in the solution, a photosensitized process starts, where Bip photoinduces the oxidation of H2Bip to Bip, and H2O2 is formed. As a consequence, the rates of H2Bip consumption and Bip formation increase as a function of irradiation time, resulting in an autocatalytic photochemical process. In this process, Bip in its triplet excited state reacts with the ground state of H2Bip. The mechanisms involved are analyzed and the biological implications of the results are discussed.
Hiroyuki Hasegawa - One of the best experts on this subject based on the ideXlab platform.
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asymmetric uptake of sepiapterin and 7 8 Dihydrobiopterin as a gateway of the salvage pathway of tetrahydrobiopterin biosynthesis from the lumenal surface of rat endothelial cells
Molecular Genetics and Metabolism, 2011Co-Authors: Akiko Ohashi, Kaori Mamada, Isao Tsuboi, Shin Aizawa, Hiroyuki HasegawaAbstract:Abstract Rat aortic endothelial cells were cultured on a porous membrane to form a monolayer sheet. They efficiently accumulated tetrahydrobiopterin (BH 4 ) by uptake of sepiapterin but did so only moderately by uptake of Dihydrobiopterin. The endothelial cell sheet preferentially took up the pterins from the apical side. Accordingly, a dense accumulation of ENT2-like immunoreactivity was visualized on the apical surface of the cell sheet. The findings suggest that vascular endothelial cells receive BH 4 precursors directly from the blood stream rather than from ablumenal tissues.
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Tetrahydrobiopterin uptake in supplemental administration: elevation of tissue tetrahydrobiopterin in mice following uptake of the exogenously oxidized product 7,8-Dihydrobiopterin and subsequent reduction by an anti-folate-sensitive process.
Journal of pharmacological sciences, 2004Co-Authors: Keiko Sawabe, Kazunori Wakasugi, Hiroyuki HasegawaAbstract:In order to increase the tissue level of tetrahydrobiopterin (BH4), supplementation with 6R-tetrahydrobiopterin (6RBH4) has been widely employed. In this work, the effectiveness of 6RBH4 was compared with 7,8-Dihydrobiopterin (7,8BH2) and sepiapterin by administration to mice. Administration of 6RBH4 was the least effective in elevating tissue BH4 levels in mice while sepiapterin was the best. In all three cases, a Dihydrobiopterin surge appeared in the blood. The appearance of the Dihydrobiopterin surge after BH4 treatment suggested that systemic oxidation of the administered BH4 had occurred before accumulation of BH4 in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH4 was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH2 to BH4. 2) When the unnatural diastereomer 6SBH4 was administered to mice, a large proportion of the recovered BH4 was in the form of the 6R-diastereomer, suggesting that this BH4 was the product of a dihydrofolate reductase process by which 7,8BH2 converts to 6RBH4. These results indicated that the exogenous BH4 was oxidized and the resultant 7,8BH2 circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH2 in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by de novo synthesis and by salvage of extracellular Dihydrobiopterin.
Antonius C F Gorren - One of the best experts on this subject based on the ideXlab platform.
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Interaction between Neuronal Nitric-Oxide Synthase and Tetrahydrobiopterin Revisited: Studies on the Nature and Mechanism of Tight Pterin Binding
2016Co-Authors: Christian L Heine, Bernd Kolesnik, Renate Schmidt, Ernst R Werner, Bernd Mayer, Antonius C F GorrenAbstract:(nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10−7−10−6 M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10−7 M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-Dihydrobiopterin or 4-amino-Dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formatio
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Interaction between Neuronal Nitric-Oxide Synthase and Tetrahydrobiopterin Revisited: Studies on the Nature and Mechanism of Tight Pterin Binding
2015Co-Authors: Christian L. Heine, Bernd Kolesnik, Renate Schmidt, Ernst R Werner, Bernd Mayer, Antonius C F GorrenAbstract:Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10–7–10–6 M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10–7 M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-Dihydrobiopterin or 4-amino-Dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry
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interaction between neuronal nitric oxide synthase and tetrahydrobiopterin revisited studies on the nature and mechanism of tight pterin binding
Biochemistry, 2014Co-Authors: Christian L Heine, Bernd Kolesnik, Renate Schmidt, Ernst R Werner, Bernd Mayer, Antonius C F GorrenAbstract:Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10(-7)-10(-6) M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10(-7) M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-Dihydrobiopterin or 4-amino-Dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.
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electrochemistry of pterin cofactors and inhibitors of nitric oxide synthase
Nitric Oxide, 2001Co-Authors: Antonius C F Gorren, Ernst R Werner, Andreas J Kungl, Kurt Schmidt, Bernd MayerAbstract:Abstract Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), but its function is not fully understood. Specifically, it is unclear whether BH4 participates directly in electron transfer. We investigated the redox properties of BH4 and several other pteridines with cyclic voltammetry and Osteryoung square wave voltammetry. BH4 was oxidized at a potential of +0.27 V vs normal hydrogen electrode (NHE); the corresponding reductive signal after the reversal of the scan direction was very small. Instead, reduction occurred at a potential of −0.16 V vs NHE; there was no corresponding oxidative signal. These two transitions were interdependent, indicating that the reductive wave at −0.16 V represented the regeneration of BH4 from its product of oxidation at +0.27 V. Similar voltammograms were obtained with tetrahydroneopterin and 6,7-dimethyltetrahydropterin, both of which can substitute for BH4 in NOS catalysis. Completely different voltammograms were obtained with 7,8-Dihydrobiopterin, sepiapterin, 2′-deoxysepiapterin, and autoxidized BH4. These 7,8-dihydropterins, which do not sustain NOS catalysis, were oxidized at much higher potentials (+0.82–1.04 V vs NHE), and appreciable reduction did not occur between +1.2 and −0.8 V, in line with the concept of a redox role for BH4 in NOS catalysis. However, the electrochemical properties of the potent pterin-site NOS inhibitor 4-amino-BH4 resembled those of BH4, whereas the active pterin cofactor 5-methyl-BH4 was not re-reduced after oxidation. We conclude that the 2-electron redox cycling of the pterin cofactor between BH4 and quinonoid Dihydrobiopterin is not essential for NO synthesis. The data are consistent with 1-electron redox cycling between BH4 and the trihydrobiopterin radical BH3•.
Seymour Kaufman - One of the best experts on this subject based on the ideXlab platform.
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The regulation of dopamine release from striatum slices by tetrahydrobiopterin and l-arginine-derived nitric oxide
Brain research, 1998Co-Authors: Li Ping Liang, Seymour KaufmanAbstract:The regulation of dopamine release by 6(R)-tetrahydrobiopterin (BH4) and l-arginine-derived nitric oxide was examined by using a method of superfusion of rat striatum slices in vitro. l-Arginine, which can produce nitric oxide (NO) through the action of NO synthase, induces a concentration-dependent increase of [3H] dopamine release in the superfusate of striatum slices. Pretreatment with inhibitors of NO synthase or with inhibitors of BH4 synthesis diminishes the increase of [3H] dopamine release mediated by arginine. This increase is almost completely restored following repletion of intracellular BH4 levels by incubation of the slices with 7, 8-Dihydrobiopterin. Adding exogenous BH4 directly to the superfusion fluid leads to a massive increase in [3H] dopamine release which can be inhibited 75% by superoxide dismutase and catalase, but is not inhibited by NG-nitro-arginine, a NO synthase inhibitor, or alpha-methyl-p-tyrosine, a tyrosine hydroxylase inhibitor. The increase of intracellular BH4 concentration by Dihydrobiopterin administration causes a small increase of dopamine release which can be partially diminished by NG-nitro-arginine or alpha-methyl-p-tyrosine. It is suggested that the increase of dopamine release stimulated by an enhancement of intracellular BH4 is dependent on its cofactor activity with NO synthase and tyrosine hydroxylase. This study has also demonstrated that BH4 is a regulator of NO-mediated dopamine release in the striatum. Published by Elsevier Science B.V.
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Reduction of Quinonoid Dihydrobiopterin to Tetrahydrobiopterin by Nitric Oxide Synthase
The Journal of biological chemistry, 1996Co-Authors: Cor F.b. Witteveen, John Giovanelli, Seymour KaufmanAbstract:Abstract Rat cerebellar nitric oxide synthase (NOS) purified from transfected human kidney cells catalyzes an NADPHdependent reduction of quinonoid Dihydrobiopterin (qBH) to tetrahydrobiopterin (BH). Reduction of qBH at 25 μM proceeds at a rate that is comparable with that of the overall reaction (citrulline synthesis) and requires calcium ions and calmodulin for optimal activity; NADH has only 10% of the activity of NADPH. The reduction rate with the quinonoid form of 6-methyldihydropterin is approximately twice that with qBH. 7,8-Dihydrobiopterin had negligible activity. Neither 7,8-Dihydrobiopterin nor BH affected the rate of qBH reduction. Reduction is inhibited by the flavoprotein inhibitor diphenyleneiodonium, whereas inhibitors of electron transfer through heme (7-nitroindazole and N-nitroarginine) stimulated the rate to a small extent. Methotrexate, which inhibits a variety of enzymes catalyzing Dihydrobiopterin reduction, did not inhibit. These studies provide the first demonstration of the reduction of qBH to BH by NOS and indicate that the reduction is catalyzed by the flavoprotein “diaphorase” activity of NOS. This activity is located on the reductase (C-terminal) domain, whereas the high affinity BH site involved in NOS activation is located on the oxygenase (N-terminal) domain. The possible significance of this reduction of qBH to the essential role of BH in NOS is discussed.
Adriana Ipiña - One of the best experts on this subject based on the ideXlab platform.
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Solar radiation exposure of Dihydrobiopterin and biopterin in aqueous solution
Solar Energy, 2014Co-Authors: Adriana Ipiña, Carolina Castaño, M. Laura Dántola, Andrés H. ThomasAbstract:Abstract Dihydrobiopterin (H2Bip) and biopterin (Bip) accumulate in the skin of patients suffering from vitiligo, whose lack of melanin causes a deficient protection against UV radiation. H2Bip and Bip undergo photooxidation upon UV irradiation and the former acts as a photosensitizer of biomolecules. The aim of this work is to study the photochemical reactions of these compounds triggered by sunlight under outdoor conditions. H2Bip and Bip in aqueous solutions were exposed to sunlight in La Plata city (34.90°S, 57.92°W) from Spring equinox to near Summer solstice and the spectral solar irradiance was recorded with a high-resolution spectrometer. The photochemical reactions were followed by HPLC and UV-Vis spectrophotometry. Upon sun exposure, excitation of H2Bip leads to the formation of dimers and to its oxidation to Bip, which, in turn, is photooxidized into 6-formylpterin (Fop). Further excitation induces the oxidation of Fop to 6-carboxypterin, which is much more photostable than Bip and Fop and then it is accumulated in the solution. Rates of reactant consumption were determined under different weather conditions and the corresponding quantum yields were also calculated. We have demonstrated that solar radiation causes significant oxidation of the pterin derivatives investigated within a few minutes, even in cloudy days. Finally, the biological implications of our results are discussed.