RESUMEN
Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M-1·s-1, (9 ± 2) × 104 M-1·s-1, and (1.6 ± 0.5) × 105 M-1·s-1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/metabolismo , Electrones , Miocardio/metabolismo , NADH Deshidrogenasa/metabolismo , Zinc/metabolismo , 2,6-Dicloroindofenol/metabolismo , Animales , Dominio Catalítico , Escherichia coli/enzimología , Peróxido de Hidrógeno/metabolismo , Cinética , Oxidación-Reducción , Superóxidos/metabolismo , Porcinos , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Sigma 1 receptor (Sig1R), a putative molecular chaperone, has emerged as a novel therapeutic target for retinal degenerative disease. Earlier studies showed that activation of Sig1R via the high-affinity ligand (+)-pentazocine ((+)-PTZ) induced profound rescue of cone photoreceptor cells in the rd10 mouse model of retinitis pigmentosa; however the mechanism of rescue is unknown. Improved cone function in (+)-PTZ-treated mice was accompanied by reduced oxidative stress and normalization of levels of NRF2, a transcription factor that activates antioxidant response elements (AREs) of hundreds of cytoprotective genes. Here, we tested the hypothesis that modulation of NRF2 is central to Sig1R-mediated cone rescue. Activation of Sig1R in 661W cone cells using (+)-PTZ induced dose-dependent increases in NRF2-ARE binding activity and NRF2 gene/protein expression, whereas silencing Sig1R significantly decreased NRF2 protein levels and increased oxidative stress, although (+)-PTZ did not disrupt NRF2-KEAP1 binding. In vivo studies were conducted to investigate whether, in the absence of NRF2, activation of Sig1R rescues cones. (+)-PTZ was administered systemically for several weeks to rd10/nrf2+/+ and rd10/nrf2-/- mice. Through post-natal day 42, cone function was significant in rd10/nrf2+/+, but minimal in rd10/nrf2-/- mice as indicated by electroretinographic recordings using natural noise stimuli, optical coherence tomography and retinal histological analyses. Immunodetection of cones was limited in (+)-PTZ-treated rd10/nrf2-/-, though considerable in (+)-PTZ-treated rd10/nrf2+/+mice. The data suggest that Sig1R-mediated cone rescue requires NRF2 and provide evidence for a previously-unrecognized relationship between these proteins.
Asunto(s)
Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Receptores sigma/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Degeneración Retiniana/terapia , Animales , Proteína 1 Asociada A ECH Tipo Kelch/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Receptores sigma/genética , Receptor Sigma-1RESUMEN
An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.
Asunto(s)
Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Línea Celular Tumoral , Genes Reporteros , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
HIF prolyl hydroxylase is a major regulator of HIF stability. Branched tail oxyquinolines have been identified as specific inhibitors of HIF prolyl hydroxylase and recently demonstrated clear benefits in various scenarios of neuronal failure. The structural optimization for branched tail oxyquinolines containing an acetamide bond has been performed in the present study using HIF1 ODD-luc reporter assay. The special attention has been paid to the length of a linker between acetamide group and phenyl ring, as well as substitutions in the phenyl ring in the other branch of the tail. The optimized version of branched tail oxyquinolines is 3-fold more potent than the original one identified before and shows a submicromolar EC50 in the reporter assay. The compounds have been studied in a "liver-on-a-chip" device to question their hepatotoxicity towards differentiated human HepaRG "hepatocytes": the absence of hepatotoxicity is observed up to 200 µM concentrations for all studied derivatives of branched tail oxyquinolines.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Prolina Dioxigenasas del Factor Inducible por Hipoxia/biosíntesis , Oxiquinolina/química , Acetamidas/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxiquinolina/farmacología , Relación Estructura-ActividadRESUMEN
Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis.
Asunto(s)
Aminoácidos Aromáticos , Mutagénesis Sitio-Dirigida , Peroxidasas/química , Peroxidasas/metabolismo , Benzotiazoles/metabolismo , Biocatálisis , Estabilidad de Enzimas , Peróxido de Hidrógeno/metabolismo , Cinética , Modelos Moleculares , Oxidación-Reducción , Peroxidasas/genética , Conformación Proteica , Replegamiento Proteico , Especificidad por Sustrato , Ácidos Sulfónicos/metabolismo , TemperaturaRESUMEN
Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 µg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.6, with 1 µM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture).
Asunto(s)
Escherichia coli/genética , Peroxidasas/química , Peroxidasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Hemina , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión , Peroxidasas/genética , Peroxidasas/aislamiento & purificación , Replegamiento Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , UreaRESUMEN
This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely ß2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Catálisis , Diseño de Fármacos , Factor 1 Inducible por Hipoxia/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Isoformas de Proteínas , Especificidad por SustratoRESUMEN
The screening of potential redox mediators for laccase was performed using homogeneous Trametes hirsuta laccase. Heterogeneous (electrochemical) and homogeneous (oxidation by laccase) reactions of the different types of the enhancers (mediators) of the enzyme were investigated. It was discovered that derivatives of phenyl-methyl-pyrazolones and benzoic acid, as well as N-hydroxynaphthalimide were efficient substrates for the laccase. The characterization of several representatives from each class was carried out using electrochemical and enzyme kinetics methods. The kinetic parameters for the oxidation of phenyl-methyl-pyrazolones and 3-(6-hylroxy)-aminobenzoic acid were comparable to those for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by the laccase, whereas the rate of enzymatic oxidation of N-hydroxynaphthalimide was sufficiently lower. Electrochemical experiments demonstrated that only oxidation of phenyl-methyl-pyrazolones and N-hydroxynaphthalimide yielded several high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, whereas derivatives of benzoic acid showed low-potential intermediate, which was not able to oxidized lignin model compound. Phenyl-methyl-pyrazolones was about 50% as effective in degrading veratryl alcohol compared to ABTS as judged from HPLC kinetic studies, whereas N-hydroxynaphthalimide showed the same efficiency as ABTS. Phenyl-methyl-pyrazolones and hydroxynaphthalimides may be of commercial interest for oxidoreductase-catalyzed biodegradation of different xenobiotics.
Asunto(s)
Benzoatos/química , Lacasa/química , Pirazolonas/química , Quinolonas/química , Basidiomycota/enzimología , Benzotiazoles , Electroquímica , Activación Enzimática , Cinética , Estructura Molecular , Oxidación-Reducción , Especificidad por Sustrato , Ácidos Sulfónicos/químicaRESUMEN
New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50 degrees C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K(4)Fe(CN)(6) were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.
Asunto(s)
Basidiomycota/enzimología , Proteínas Fúngicas/química , Lacasa/química , Sitios de Unión , Especificidad por SustratoRESUMEN
Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.
Asunto(s)
Escherichia coli/genética , Peroxidasas/biosíntesis , Peroxidasas/genética , Peroxidasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Clonación Molecular , Cuerpos de Inclusión/enzimología , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Significant conformational differences between native and recombinant horseradish peroxidase have been shown by tritium planigraphy, which includes a method of thermal activation of tritium followed by amino acid analysis of the protein preparation. Comparison of radioactivity distribution among the amino acid residues with the theoretical (calculated) accessibility shows that the recombinant enzyme is characterized by high hydrophobicity and compactness of folding. The protective role of oligosaccharides in native enzyme has been confirmed. An unexpected result of the study is a finding on high accessibility of a catalytic histidine residue in solution. An effect of low dose (3 Gy) of irradiation on the accessibility of amino acid residues has been unequivocally demonstrated. The data can be interpreted as swelling of the compact folding and increase in the surface hydrophilicity of the recombinant enzyme. In the case of native enzyme, irradiation does not cause remarkable changes in the accessibility of amino acid residues indicating the possible extensive radical modification of the native enzyme in the life-course of the cell. The catalytic histidine is an exception. It becomes inaccessible after the enzyme irradiation, while its accessibility in the recombinant enzyme increases. An additional observation of a 5-fold decrease in the rate constant towards hydrogen peroxide points to the destructive effect of irradiation on the hydrogen bond network in the distal domain of the native enzyme molecule and partial collapse of the active site pocket.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Proteínas Recombinantes/química , Aminoácidos/análisis , Estabilidad de Enzimas/efectos de la radiación , Rayos gamma , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/aislamiento & purificación , Peroxidasa de Rábano Silvestre/efectos de la radiación , Conformación Proteica/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/efectos de la radiación , TritioRESUMEN
The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3'-Sulfophenyl)-3- methylpyrazolone-5 was about 30-40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commercial interest for oxidoreductase-catalyzed biodegradation of organic compounds.
Asunto(s)
Lacasa/metabolismo , Basidiomycota/enzimología , Benzotiazoles , Alcoholes Bencílicos/química , Alcoholes Bencílicos/metabolismo , Cromatografía Líquida de Alta Presión , Electroquímica , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Lacasa/aislamiento & purificación , Peso Molecular , Oxidación-Reducción , Pirazoles/química , Pirazoles/metabolismo , Espectrofotometría , Especificidad por Sustrato , Ácidos Sulfónicos/química , Ácidos Sulfónicos/metabolismoRESUMEN
A quantitative approach for estimation of the non-enzymatic interaction between ammonium 2,2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation product and a poorly oxidized substrate was developed using a system including tobacco peroxidase, a mediator substrate (ABTS), and a second substrate. The approach is based on the establishment of a pseudo-steady-state concentration of the ABTS oxidation product in the course of co-oxidation with a poor substrate. A mathematical description of the experimental curve shape has been proposed to linearize the kinetic data and estimate the rate constant for such non-enzymatic interaction. The rate constants calculated from the steady-state kinetics for the non-enzymatic interaction of ABTS oxidation product with phenol and resorcinol were 360 +/- 40 and 770 +/- 60 M(-1).sec(-1), respectively. The values obtained have the same order of magnitude as the rate constant for ABTS oxidation product interaction with veratryl alcohol, calculated from electrochemical measurements (170 M(-1).sec(-1)) by Donal Leech's group. However, the kinetic curves for co-oxidation of ABTS and veratryl alcohol catalyzed by tobacco peroxidase exhibit a pronounced lag-period, which either points to the high rate of the non-enzymatic interaction between ABTS oxidation product and veratryl alcohol and thus, contradicts the electrochemical calculations, or indicates an enzymatic nature of the co-oxidation phenomenon in this particular case.
Asunto(s)
Nicotiana/enzimología , Peroxidasa/metabolismo , Alcoholes/metabolismo , Benzotiazoles , Catálisis/efectos de los fármacos , Cinética , Fenol/farmacología , Resorcinoles/metabolismo , Ácidos Sulfónicos/química , Ácidos Sulfónicos/metabolismoRESUMEN
Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.
Asunto(s)
Peroxidasa de Rábano Silvestre/metabolismo , Triptófano/química , Catálisis , Estabilidad de Enzimas , Peroxidasa de Rábano Silvestre/química , Concentración de Iones de Hidrógeno , Cinética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.
Asunto(s)
Técnicas Biosensibles/métodos , Peroxidasas , Arachis/enzimología , Electroquímica , Transporte de Electrón , Peroxidasa de Rábano Silvestre/metabolismo , Cinética , Peroxidasas/metabolismo , Solanaceae/enzimologíaRESUMEN
The application of a newly isolated transgenic tobacco peroxidase (TOP) as a chemiluminescent label for immunoassay purposes is described for the first time. The enzyme has been oxidized with m-periodate and subsequently coupled to the model compound 2,4-dichlorophenoxyacetic acid (2,4-D) using a carbodiimide method. As compared to the native horseradish peroxidase used in control experiments, the TOP enzyme showed significantly higher efficiency of coupling to the antigen and no loss of the specific activity was observed. The obtained 2,4-D-TOP conjugate demonstrated unique properties in chemiluminescent detection. The latter allowed the minimization of the conjugate concentration due to the superior chemiluminescent activity of the enzyme. A highly sensitive capillary chemiluminescent immunoassay using the 2,4-D-TOP conjugate as labeled competitor is reported. Direct competitive ELISA has been performed using a specific monoclonal antibody immobilized onto the sol-gel treated glass capillary surface. A modified photomultiplier tube with a special holder for a capillary was used for the resulting chemiluminescent signal detection. The typical standard calibration curve for the 2,4-D pesticide detection is linear between 30 pg and 500 ng/mL.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/análisis , Herbicidas/análisis , Técnicas para Inmunoenzimas , Mediciones Luminiscentes , Sensibilidad y EspecificidadRESUMEN
The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex.
Asunto(s)
Sustitución de Aminoácidos , Peroxidasa de Rábano Silvestre/metabolismo , Triptófano/química , Secuencia de Aminoácidos , Catálisis , Electrones , Activación Enzimática/efectos de la radiación , Estabilidad de Enzimas/efectos de la radiación , Escherichia coli/genética , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/genética , Peroxidasa de Rábano Silvestre/aislamiento & purificación , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión , Mediciones Luminiscentes , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Plantas/enzimología , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , Triptófano/genéticaRESUMEN
Indole-3-acetic acid (IAA) can be oxidized via two mechanisms: a conventional hydrogen-peroxide-dependent pathway, and one that is hydrogen-peroxide-independent and requires oxygen. It has been shown here for the first time that only plant peroxidases are able to catalyse the reaction of IAA oxidation with molecular oxygen. Cytochrome c peroxidase (CcP), fungal peroxidases (manganese-dependent peroxidase, lignin peroxidase and Arthromyces ramosus peroxidase) and microperoxidase were essentially inactive towards IAA in the absence of added H2O2. An analysis of amino acid sequences allowed five structurally similar fragments to be identified in auxin-binding proteins and plant peroxidases. The corresponding fragments in CcP and fungal peroxidases showed no similarity with auxin-binding proteins. Five structurally similar fragments form a subdomain including the catalytic centre and two residues highly conserved among 'classical' plant peroxidases only, namely His-40 and Trp-117. The subdomain identified above with the two residues might be responsible for the oxidation of the physiological substrate of classical plant peroxidases, IAA.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Oxidantes/metabolismo , Oxígeno/metabolismo , Peroxidasas/metabolismo , Reguladores del Crecimiento de las Plantas , Proteínas de Plantas , Plantas/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Secuencia Conservada/genética , Secuencia Conservada/fisiología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxidasas/química , Conformación Proteica , Receptores de Superficie Celular , Relación Estructura-ActividadRESUMEN
An amperometric flow system combined with a glucose oxidase-mutarotase reactor was optimized and used to determine aromatic amines and phenols using peroxidase-modified graphite electrodes. An increase in currents upon injection of the analyzed substrate was shown to be approximated by a Michaelis-Menten type dependence. The detection limit was calculated as 3 times the noise, and the sensitivity was calculated as Imax/K(m)app. Commercially available horseradish peroxidase was compared with tobacco anionic and peanut cationic peroxidases for determination of aromatic amines and phenols. Detection limits of 10 nM for determination of o-aminophenol and o- and p-phenylenediamine achieved with a tobacco peroxidase-modified electrode give a promise for further improvements in sensitivities and detection limits of biosensors.
Asunto(s)
Compuestos de Anilina/análisis , Técnicas Biosensibles , Fenoles/análisis , Animales , Arachis/enzimología , Aspergillus niger/enzimología , Carbohidrato Epimerasas , Análisis de Inyección de Flujo , Glucosa Oxidasa , Peroxidasa de Rábano Silvestre , Riñón/enzimología , Plantas Modificadas Genéticamente , Plantas Tóxicas , Estereoisomerismo , Porcinos , Nicotiana/enzimologíaRESUMEN
Application of computer methods allowed us to demonstrate that plant peroxidases and auxin-binding proteins contain structurally similar fragments. The mapping of the fragments was done using a model structure of horseradish peroxidase. Five of six structurally similar fragments belong to the distal domain and form a subdomain in plant peroxidases that includes the distal heme-coordinating sequence, LHFHDC (amino acid residues 39-44 in horseradish peroxidase). The existence of a substrate-binding site for indole-3-acetic acid in the distal subdomain comprising helices A (whole), B (middle), C (beginning), and D (whole) and the loop between helices D and D' is discussed.