RESUMEN

We oxidized histidine residues in monoclonal antibody drugs of immunoglobulin gamma 1 (IgG1) using ultraviolet C irradiation (UVC: 200-280 nm), which is known to be potent for sterilization or disinfection. Among the reaction products, we identified asparagine and aspartic acid by mass spectrometry. In the photo-induced oxidation of histidine in angiotensin II, 18O atoms from H218O in the solvent were incorporated only into aspartic acid but not into asparagine. This suggests that UVC irradiation generates singlet oxygen and induces [2 + 2] cycloaddition to form a dioxetane involving the imidazole Cγ - Cδ2 bond of histidine, followed by ring-opening in the manner of further photo-induced retro [2 + 2] cycloaddition. This yields an equilibrium mixture of two keto-imines, which can be the precursors to aspartic acid and asparagine. The photo-oxidation appears to occur preferentially for histidine residues with lower pKa values in IgG1. We thus conclude that the damage due to UVC photo-oxidation of histidine residues can be avoided in acidic conditions where the imidazole ring is protonated.

Asunto(s)

Anticuerpos Monoclonales/química , Histidina/química , Inmunoglobulina G/química , Oxígeno Singlete/química , Angiotensina II/química , Anticuerpos Monoclonales/efectos de la radiación , Histidina/efectos de la radiación , Humanos , Imidazoles/química , Inmunoglobulina G/efectos de la radiación , Espectrometría de Masas , Oxidación-Reducción/efectos de la radiación , Rayos Ultravioleta

RESUMEN

PURPOSE: Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His. METHODS: A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase. RESULTS: The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking. CONCLUSIONS: In nucleophilic buffers, light can induce covalently-linked adducts to His residues.

Asunto(s)

Reactivos de Enlaces Cruzados/efectos de la radiación , Histidina/efectos de la radiación , Luz/efectos adversos , Reactivos de Enlaces Cruzados/química , Desoxirribonucleasas/química , Desoxirribonucleasas/efectos de la radiación , Histidina/química , Oxidación-Reducción/efectos de la radiación , Desnaturalización Proteica/efectos de la radiación , Trometamina/química

RESUMEN

The effects of γ-radiation sterilization on the parenteral excipient l-histidine were analysed by means of EPR spectroscopy. The irradiation process was found to induce the formation of a deamination radical which was persistent in the solid state. The nature and reactivity of the radicals following dissolution in water was evaluated using spin-trapping EPR experiments. The deamination radical was found to regenerate in solution in the presence of trace metals, potentially leading to radical induced degradation reactions occurring up to an hour after the dissolution process. Understanding this process is significant for the improved design of parental pharmaceutical formulations in which unwanted radical reactions after γ-radiation sterilization could lead to degradation of active ingredients.

Asunto(s)

Excipientes/efectos de la radiación , Radicales Libres/química , Rayos gamma , Histidina/efectos de la radiación , Esterilización/métodos , Espectroscopía de Resonancia por Spin del Electrón , Excipientes/química , Histidina/química , Polvos

RESUMEN

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D. I. Photochem. Photobiol. Sci. 2012 , 11 , 38 - 53 ). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here, we used rhDNase, which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photoreactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.

Asunto(s)

Desoxirribonucleasa I/efectos de la radiación , Histidina/efectos de la radiación , Multimerización de Proteína/efectos de la radiación , Desoxirribonucleasa I/química , Histidina/química , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de la radiación , Rayos Ultravioleta , Agua/química

RESUMEN

Morphology-controlled synthesis of CdS can significantly enhance the efficiency of its photocatalytic hydrogen production. In this study, a novel three-dimensional (3D) flower-like CdS is synthesized via a facile template-free hydrothermal process using Cd(NO3)2•4H2O and thiourea as precursors and L-Histidine as a chelating agent. The morphology, crystal phase, and photoelectrochemical performance of the flower-like CdS and pure CdS nanocrystals are carefully investigated via various characterizations. Superior photocatalytic activity relative to that of pure CdS is observed on the flower-like CdS photocatalyst under visible light irradiation, which is nearly 13 times of pure CdS. On the basis of the results from SEM studies and our analysis, a growth mechanism of flower-like CdS is proposed by capturing the shape evolution. The imidazole ring of L-Histidine captures the Cd ions from the solution, and prevents the growth of the CdS nanoparticles. Furthermore, the photocatalytic contrast experiments illustrate that the as-synthesized flower-like CdS with L-Histidine is more stable than CdS without L-Histidine in the hydrogen generation.

Asunto(s)

Compuestos de Cadmio/química , Histidina/química , Hidrógeno/química , Hidrógeno/aislamiento & purificación , Nanopartículas del Metal/química , Fotoquímica/métodos , Sulfuros/química , Adsorción , Compuestos de Cadmio/efectos de la radiación , Catálisis , Histidina/efectos de la radiación , Luz , Ensayo de Materiales , Nanopartículas del Metal/efectos de la radiación , Nanopartículas del Metal/ultraestructura , Tamaño de la Partícula , Dosis de Radiación , Sulfuros/efectos de la radiación

RESUMEN

Photochemical transformations greatly affect the stability and fate of amino acids (AAs) in sunlit aquatic ecosystems. Whereas the direct phototransformation of dissolved AAs is well investigated, their indirect photolysis in the presence of chromophoric dissolved organic matter (CDOM) is poorly understood. In aquatic systems, CDOM may act both as sorbent for AAs and as photosensitizer, creating microenvironments with high concentrations of photochemically produced reactive intermediates, such as singlet oxygen (1O2). This study provides a systematic investigation of the indirect photochemical transformation of histidine (His) and histamine by 1O2 in solutions containing CDOM as a function of solution pH. Both His and histamine showed pH-dependent enhanced phototransformation in the CDOM systems as compared to systems in which model, low-molecular-weight 1O2 sensitizers were used. Enhanced reactivity resulted from sorption of His and histamine to CDOM and thus exposure to elevated 1O2 concentrations in the CDOM microenvironment. The extent of reactivity enhancement depended on solution pH via its effects on the protonation state of His, histamine, and CDOM. Sorption-enhanced reactivity was independently supported by depressed rate enhancements in the presence of a cosorbate that competitively displaced His and histamine from CDOM. Incorporating sorption and photochemical transformation processes into a reaction rate prediction model improved the description of the abiotic photochemical transformation rates of His in the presence of CDOM.

Asunto(s)

Histamina/efectos de la radiación , Histidina/efectos de la radiación , Procesos Fotoquímicos , Adsorción , Ambiente , Concentración de Iones de Hidrógeno , Cinética , Modelos Teóricos , Oxígeno Singlete/química , Soluciones , Temperatura

RESUMEN

The anticancer agents cisplatin and carboplatin bind to histidine in a protein. This crystal structure study at data-collection temperatures of 100 and 300 K examines their relative binding affinities to a histidine side chain and the effect of a high X-ray radiation dose of up to ∼1.8 MGy on the stability of the subsequent protein-Pt adducts. Cisplatin binding is visible at the histidine residue, but carboplatin binding is not. Five refined X-ray crystal structures are presented: one at 100 K as a reference and four at 300 K. The diffraction resolutions are 1.8, 2.0, 2.8, 2.9 and 3.5 Å.

Asunto(s)

Carboplatino/química , Cisplatino/química , Histidina/química , Muramidasa/química , Rayos X , Animales , Carboplatino/metabolismo , Pollos , Cisplatino/metabolismo , Cisplatino/efectos de la radiación , Cristalización , Cristalografía por Rayos X , Histidina/metabolismo , Histidina/efectos de la radiación , Muramidasa/metabolismo , Unión Proteica/efectos de la radiación , Temperatura , Rayos X/efectos adversos

RESUMEN

Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.

Asunto(s)

Histidina/análogos & derivados , Lactalbúmina/química , Riboflavina/química , Tirosina/análogos & derivados , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/análisis , Radicales Libres/química , Histidina/análisis , Histidina/química , Histidina/efectos de la radiación , Lactalbúmina/efectos de la radiación , Luz , Oxidación-Reducción , Riboflavina/efectos de la radiación , Espectrometría de Masa por Ionización de Electrospray , Detección de Spin , Espectrometría de Masas en Tándem , Tirosina/análisis , Tirosina/química , Tirosina/efectos de la radiación

RESUMEN

Using amino-acid histidine as chelating agent, CdS nanoparticles have been synthesized by sonochemical method. It is found that by varying the ultrasonic irradiation time, we can tune the band gap and particle size of CdS nanoparticles. The imidazole ring of histidine captures the Cd ions from the solution, and prevents the growth of the CdS nanoparticles. The deviation in the linear relation in between cube of radius of nanoparticles and ultrasonic irradiation time confirms the growth of CdS nanoparticles occur via two process; one is the diffusion process of the reactants as well as reaction at the surface of the crystallite. CdS nanoparticles synthesized using histidine as organic chelating agent have band edge emission at approximately 481 nm and have greater photoluminescence intensity with blue-shift to higher energy due to typical quantum confinement effect.

Asunto(s)

Compuestos de Cadmio/síntesis química , Compuestos de Cadmio/efectos de la radiación , Histidina/química , Sustancias Luminiscentes/síntesis química , Sustancias Luminiscentes/efectos de la radiación , Nanopartículas/química , Compuestos de Selenio/síntesis química , Compuestos de Selenio/efectos de la radiación , Sonicación/métodos , Quelantes/química , Quelantes/efectos de la radiación , Cristalización/métodos , Histidina/efectos de la radiación , Ensayo de Materiales , Nanopartículas/efectos de la radiación , Nanopartículas/ultraestructura , Nanotecnología/métodos , Tamaño de la Partícula , Dosis de Radiación

RESUMEN

Mechanisms of flavin-mediated photooxidation of electron-rich amino acids tryptophan and histidine were investigated for aqueous solutions. Indole, representing the tryptophan side chain in proteins, reacted at nearly diffusion controlled rates (k approximately 2.7 x 10(9) L mol(-1) s(-1) at 293 K) with the triplet-excited flavin state, but reactions of imidazole (and histidine) were significantly slower (k < 2.0 x 10(8) L mol(-1) s(-1)) as determined by laser flash photolysis. Oxidation rates of derivates were invariably susceptible to electronic factors affecting incipient radical cation stability, while no primary kinetic hydrogen/deuterium isotope effect was observed for imidazole. Thus reaction by electron transfer was proposed in contrast to a direct hydrogen abstraction. Unlike indole compounds, imidazole derivatives suffered from the presence of a basic imino nitrogen (=N-), which caused the rate constant of histidine free base (k approximately 1.8 x 10(8) L mol(-1) s(-1)) to drop considerably upon protonation. Complexation of the imino nitrogen with transition metals provoked changes in reactivity, as rate constants decreased after addition of Zn(2+) (k of 4-methylimidazole, as histidine model, decreased from 9.0 x 10(8) L mol(-1) s(-1) in the absence to 4.1 x 10(8) L mol(-1) s(-1) in the presence of ZnCl(2)). The pyrrole nitrogen (-NH-) was not directly involved in complexation reactions, but its electron density increased upon interaction with hydrogen bond-accepting anions and resulted in higher rate constants (k of 4-methylimidazole increased from 9.0 x 10(8) L mol(-1) s(-1) to 2.0 x 10(9) L mol(-1) s(-1) after addition of NaOAc). The high rate constants were in agreement with a large thermodynamical driving force, as calculated from oxidation peak potentials determined electrochemically. After oxidation, resulting radical cations were readily deprotonated and trapped by 2-methyl-2-nitrosopropane, as detected by electron paramagnetic resonance spectroscopy. Indole-derived spin adducts were attributed to selective trapping of C(3)-centered radicals, whereas spin adducts with imidazole-derivatives arose from both carbon and nitrogen-centered imidazolyl radicals.

Asunto(s)

Flavinas/química , Histidina/efectos de la radiación , Luz , Fotólisis , Triptófano/efectos de la radiación , Histidina/química , Oxidación-Reducción , Triptófano/química

RESUMEN

Quantum yields of photodecomposition and triplet state formation under aerobic and anaerobic conditions are determined for kynurenine (KN), 3-hydroxykynurenine (3OHKN), xanthurenic acid (XAN), and kynurenine adducts of glutathione (GSH-KN), cysteine (Cys-KN), histidine (His-KN), and lysine (Lys-KN) in aqueous solutions. The highest yields of anaerobic photodecomposition were obtained for GSH-KN and His-KN adducts, which correlates with the highest triplet yields for these compounds. In aerobic conditions, the photodecomposition yields for all compounds under study increase; the highest decomposition rates were observed for His-KN and 3OHKN. The fast decomposition of the latter is attributed to the dark autoxidation of the starting compound.

Asunto(s)

Quinurenina/efectos de la radiación , Fotólisis , Rayos Ultravioleta , Anaerobiosis , Cisteína/química , Cisteína/efectos de la radiación , Glutatión/química , Glutatión/efectos de la radiación , Histidina/química , Histidina/efectos de la radiación , Quinurenina/química , Teoría Cuántica , Dispersión de Radiación , Factores de Tiempo

RESUMEN

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.

Asunto(s)

Histidina/química , Magnetismo , Nanoestructuras/química , Nanoestructuras/ultraestructura , Ácido Nitrilotriacético/química , Fosfolípidos/química , Proteínas Recombinantes/química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/efectos de la radiación , Histidina/efectos de la radiación , Micelas , Movimiento (Física) , Nanoestructuras/efectos de la radiación , Proteínas Recombinantes/efectos de la radiación , Proteínas Recombinantes/ultraestructura

RESUMEN

The photobiological properties of 6-methoxy-2-naphthylacetic acid (6-MNAA) were studied using a variety of in vitro phototoxicity assays: photohemolysis, photoperoxidation of linoleic acid, photosensitized degradation of histidine and thymine and the Candida phototoxicity test. 6-MNAA was phototoxic in vitro. 6-MNAA reduced nitro blue tetrazolium (NBT) when irradiated with lambda > or = 300 nm in deoxygenated aqueous buffer solution (pH 7.4). NBT can be reduced by reaction with the excited state of 6-MNAA subject to interference with molecular oxygen. The photohemolysis rate was inhibited by the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO), sodium azide (NaN3) and reduced glutathione (GSH). Photoperoxidation of linoleic acid and photosensitized degradation of histidine and thymine were significantly inhibited by sodium azide and reduced glutathione. 6-MNAA was phototoxic to C. albicans, C. lipolytica and C. tropicalis. A mechanism involving singlet oxygen, radicals, and electron transfer reactions is suggested for the observed phototoxicity.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacocinética , Butanonas/farmacocinética , Dermatitis Fototóxica , Ácidos Naftalenoacéticos/farmacología , Fármacos Fotosensibilizantes/farmacología , Aerobiosis , Anaerobiosis , Candida/efectos de los fármacos , Candida/crecimiento & desarrollo , Candida/efectos de la radiación , ADN/efectos de los fármacos , ADN/efectos de la radiación , Eritrocitos/efectos de los fármacos , Eritrocitos/efectos de la radiación , Hemólisis/efectos de los fármacos , Hemólisis/efectos de la radiación , Histidina/química , Histidina/efectos de la radiación , Humanos , Técnicas In Vitro , Luz , Ácido Linoleico/química , Ácido Linoleico/efectos de la radiación , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Nabumetona , Nitroazul de Tetrazolio/química , Nitroazul de Tetrazolio/efectos de la radiación , Fotoquímica , Timina/química , Timina/efectos de la radiación

RESUMEN

Procedures for the vacuum deposition of thin histidine films on polycrystalline Au(111) and their characterization with high-resolution synchrotron-radiation-based photoelectron spectroscopy are reported. The chemical form of histidine (anionic vs zwitterionic) and the nature of its interactions with the substrate (strong ionic-covalent vs weak van der Waals bonding) in mono- and multilayer films are analyzed. It is shown that water adsorption on a pre-prepared histidine film at 100 K results in protonation of histidine molecules and partial formation of hydroxyl anions. These chemical effects are carefully differentiated from spectral changes associated with radiation damage of the histidine films.

Asunto(s)

Oro/química , Histidina/química , Membranas Artificiales , Sincrotrones , Adsorción , Histidina/efectos de la radiación , Concentración de Iones de Hidrógeno , Estructura Molecular , Fotones , Sensibilidad y Especificidad , Análisis Espectral/métodos , Propiedades de Superficie , Agua/química , Rayos X

RESUMEN

Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examining protein-protein interactions in solution. Hydroxyl radicals generated by radiolysis or chemical methods efficiently react with many amino acid residue side chains, including the aromatic and sulfur-containing residues along with proline and leucine, generating stable oxidation products that are valuable probes for examining protein structure. In this study, we examine the radiolytic oxidation chemistry of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidation products. Model peptides containing arginine, histidine, and lysine were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The rates of oxidation and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidation, giving rise to a characteristic product with a 43 Da mass reduction as a result of the loss of guanidino group and conversion to gamma-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidation studies. Histidine was oxidized to generate a mixture of products with characteristic mass changes primarily involving rupture of and addition to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.

Asunto(s)

Aminoácidos/química , Aminoácidos/efectos de la radiación , Péptidos/química , Péptidos/efectos de la radiación , Proteínas/química , Proteínas/efectos de la radiación , Aminoácidos/análisis , Arginina/análisis , Arginina/química , Arginina/efectos de la radiación , Dióxido de Carbono/química , Rayos gamma , Histidina/análisis , Histidina/química , Histidina/efectos de la radiación , Lisina/análisis , Lisina/química , Lisina/efectos de la radiación , Estructura Molecular , Oxidación-Reducción/efectos de la radiación , Péptidos/análisis , Unión Proteica , Proteínas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Rayos X

RESUMEN

Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.

Asunto(s)

Membrana Eritrocítica/efectos de los fármacos , Indoles/efectos adversos , Compuestos Organometálicos/efectos adversos , Fotoquimioterapia/efectos adversos , Pirimidinonas/efectos adversos , Membrana Eritrocítica/efectos de la radiación , Eritrocitos/efectos de los fármacos , Eritrocitos/efectos de la radiación , Histidina/efectos de los fármacos , Histidina/efectos de la radiación , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Fotobiología , Fármacos Fotosensibilizantes/efectos adversos , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/efectos de la radiación

RESUMEN

A new approach to the study of the interaction of amino acid side chains with photoreactive aryl azides was proposed. This approach was based on the drawing together of the reacting groups by the attachment of the reacting compounds to complementary oligonucleotides. Cystamine, histamine, and 1,6-hexamethylenediamine mimicking the cystine, histidine, and lysine residues, respectively, were attached to the 3'-terminal phosphate of the oligonucleotide GGTATCp through a phosphamide bond and used as the targets for photomodification. Derivatives of the oligonucleotide pGATACCAA with the fragment N3C6H4NH- attached directly to its 5'-end by a phosphamide bond or through the spacer -(CH2)nNH- (where n is 2, 4, and 6) were used as photoreagents. Their derivatives containing the same spacer and the N3C6F4CO-NH(CH2)3NH- or 2-N3,5-NO2-C6H3CO-NH(CH2)3NH- residues were also used. The duplexes were photomodified by irradiation with 300-350 nm wavelength light. The maximal yields of the photo-cross-linking were from 22 to 68%. The reagents containing p-azidoaniline residue were found to be the most effective toward the targets. The maximum yields of the photomodification products modeling the side chains of cysteine and lysine were found to vary from 40 to 67% and to depend on the length and the structure of the spacers used. The duplex with the target bearing the imidazole residue (the histidine model) manifested a yield decreased to 25%. This fact was in a good agreement with the data of computer modeling that indicated an unfavorable mutual displacement of the imidazole residue and the photoreactive group.

Asunto(s)

Aminoácidos/química , Azidas/química , Rayos Ultravioleta , Aminoácidos/efectos de la radiación , Azidas/efectos de la radiación , Cistamina/química , Cistamina/efectos de la radiación , Cisteína/química , Cisteína/efectos de la radiación , Diaminas/química , Diaminas/efectos de la radiación , Electroforesis en Gel de Poliacrilamida , Histamina/química , Histamina/efectos de la radiación , Histidina/química , Histidina/efectos de la radiación , Imidazoles/química , Imidazoles/efectos de la radiación , Lisina/química , Lisina/efectos de la radiación , Modelos Moleculares , Oligonucleótidos/química , Oligonucleótidos/efectos de la radiación

RESUMEN

Near-ultraviolet (NUV) light (280-400 nm) has a variety of effects on biological systems; these effects are increased, often synergistically, in the presence of sensitizers. A variety of both man-made and naturally occurring sensitizers have been identified, but their precise roles and relative contributions to cellular damage are not yet fully established. DNA seems to be a major target and a variety of types of damage have been observed. In this report we present evidence that histidine can also act as a sensitizer of NUV. Upon NUV photolysis a variety of reactive oxygen species, including superoxide anions, hydroxyl radicals and hydrogen peroxide, are produced as determined by the effects of various scavengers. pH influences the reaction, alkaline media being most effective, as has previously been reported for the photolysis of H2O2, tyrosine, phenylalanine and tryptophan. Exposure of phage T7 to a combination of histidine and NUV leads to synergistic inactivation and scavengers of O2.-, .OH and H2O2 reduce this effect. These results point to a possible involvement of sunlight-induced histidine photolysis in cellular damage. The fact that photolysis is maximal at high pH indicates that biological effects are likely to be highly localized, e.g., at enzyme active sites.

Asunto(s)

Bacteriófago T7/efectos de la radiación , Histidina/farmacología , Histidina/efectos de la radiación , Especies Reactivas de Oxígeno , Rayos Ultravioleta , Bacteriófago T7/efectos de los fármacos , Daño del ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Escherichia coli/virología , Depuradores de Radicales Libres , Histidina/química , Peróxido de Hidrógeno/química , Radical Hidroxilo/química , Fotólisis , Luz Solar , Superóxidos/química

RESUMEN

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg2+ and Mn2+ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37 degrees C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10 degrees C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas de Escherichia coli , Escherichia coli/enzimología , Guanosina Trifosfato/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Clonación Molecular , Colicinas/genética , Colicinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/efectos de la radiación , Guanosina Trifosfato/efectos de la radiación , Histidina/genética , Histidina/efectos de la radiación , Cinética , Magnesio/metabolismo , Manganeso/metabolismo , Plásmidos , Mutación Puntual , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Recombinación Genética , Rayos Ultravioleta

RESUMEN

UV-irradiation of human serum albumin, tryptophan, and histidine resulted in products formation showing antiradical activity, as detected by increased latent period in development of luminol photochemiluminescence. UV-irradiation of ascorbic acid decreased its antiradical activity. Under UV-illumination, antiradical activity of blood plasma decreased rapidly followed by a gradual increase of antiradical activity. Apparently, the former effect (decrease of antiradical activity) is a result of photolysis of natural blood antioxidants, while subsequent increase of antiradical activity is a consequence of the accumulation of plasma protein photolysis products.

Asunto(s)

Antioxidantes , Sangre/efectos de la radiación , Rayos Ultravioleta , Radicales Libres , Histidina/efectos de la radiación , Humanos , Técnicas In Vitro , Mediciones Luminiscentes , Fotoquímica , Albúmina Sérica/efectos de la radiación , Triptófano/efectos de la radiación