RESUMO
3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO) derived from the AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals (f) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction (fri) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction (fT) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes (fsyn) and homogenates (fhom), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO. Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.
Assuntos
Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Peróxidos/metabolismo , Triptofano/farmacologia , ortoaminobenzoatos/farmacologia , Álcoois/metabolismo , Amidinas/farmacologia , Animais , Antioxidantes/farmacologia , Feminino , Cinética , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Oxirredução/efeitos dos fármacos , RatosRESUMO
The osmotic condition modulates the properties of liposomes, particularly those related to their stability and response to external agents such as membrane-active proteins or peptides. In a previous work, we have demonstrated that an osmotic shock can increase, per se, water influx/efflux and the exit of the fluorophore calcein entrapped in the aqueous pool of dipalmitoylphosphatidylcholine (DPPC) and DPPC:sphingomyelin (SM) large unilamellar vesicles (LUVs), suggesting a loss of integrity of the liposome bilayer. In the present work, we have extended our study in order to assess how an osmotic imbalance prior to or synchronous with the addition of a recombinant variant of the pore-forming toxin sticholysin I (rSt I) modifies its pore forming capacity in DPPC and DPPC:SM (1:1) LUVs. Our results conclusively show the capacity of hypotonic gradients to improve the pore forming capacity of rSt I molecules, even in pure DPPC liposomes, rendering pore-formation less dependent on the presence of sphyngomyelin. In fact, non-active toxins in DPPC liposomes become active by a hypotonic imbalance in a similar way to those containing SM as a second component.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Pressão Osmótica , Proteínas Citotóxicas Formadoras de Poros/química , Compostos Orgânicos/químicaRESUMO
DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Pressão Osmótica , Esfingomielinas/química , Lipossomas Unilamelares/química , Animais , Anisotropia , Encéfalo , Fluoresceínas/química , Polarização de Fluorescência , Suínos , Água/químicaRESUMO
During the last decades the ORAC (Oxygen Radical Absorbance Capacity) assay has been widely employed to evaluate the in vitro antioxidant capacity of polyphenol-rich fruits, vegetables and beverages. The method employs fluorescein (FLH) as target molecule and AAPH (2,2'-azo-bis(2-amidinopropane)dihydrochloride) as the source of peroxyl radicals (ROOâ¢). The protection of FLH, afforded by antioxidants (XH), is often characterized by kinetic profiles with clear lag times (LT), which are directly associated with the stoichiometry (n) of the XH-ROO⢠reaction. However, even for simple phenolic compounds, the LT measured imply large n values (defined as the number of ROO⢠moles trapped by each antioxidant molecule) which cannot be explained by a simple reaction mechanism. Nonetheless, they can be explained when considering the formation of alkoxyl radicals (ROâ¢) from the recombination of two AAPH-derived ROOâ¢. In the present work, we provide kinetic data showing that, in the zero order kinetic limit of FLH consumption, there is a low reaction rate incompatible with total trapping of ROOâ¢. Thus, the consumption of FLH should be mostly related to its reaction with ROâ¢. In addition, we present data regarding the assumption that in competitive measurements, the LT is due to efficient trapping of the ROO⢠by the added phenols, leading to high n values (1.7 to 23) for mono and polyphenols. These values are not in agreement with kinetic studies of the antioxidant consumption mediated by the presence of AAPH carried out by HPLC-DAD technique, which imply a competition by ROâ¢. The results suggest that the use of FLH as probe at low concentrations give, for several antioxidants, ORAC values mainly related to their reaction towards RO⢠radicals instead of primary ROOâ¢radicals.
RESUMO
In the present work, we analyze the effect of incorporation of the nonanol family (e.g., 1-Nonanol (1-N), 5-Nonanol (5-N), and 2,6-Dimethyl-4-Heptanol (2,6-DH)) into DPPC LUVs in the presence of different gramicidin concentrations. The principal aim of this work is to study the effect of alkanols solubilization on the physicochemical properties of lipid bilayers in the presence of peptide trans-membrane channels, that is, the effects of nonanol family in the interface of lipid-peptide region, considering that the study provides the analysis of a ternary system by direct excitation as well as by Fluorescence Resonance Energy Transfer. Fluorescence measurements were carried out at 20°C after direct excitation of the extrinsic probe or by Fluorescence Resonance Energy Transfer (FRET) from the tryptophan group of gramicidin. Alkanol incorporation decreases with increasing gramicidin content and branching of the additives. 1-N generates most important changes in the inner part of the bilayer, where it produces an increase in bulk acyl chain mobility. Similarly, 1-N significantly modifies the properties of the hydrophilic-hydrophobic interface region sensed by Laurdan, increasing the polarity of the probe microenvironment and/or increasing the relaxation time of interfacial water molecules. On the other hand, 1-N produces a decrease in PDA fluorescence lifetime, a result that can be explained by a significant amount of water entrance to the inner part of the bilayer. The same behavior was observed when pseudo-first-order quenching rate constants by oxygen were measured. 1-N produces an increase in mobility/solubility of the oxygen in the lipid membrane, an effect that is more noticeable in the deep region of the bilayer sensed by PDA, in the absence and in the presence of 2 mol% of Gr. 1-N incorporation produces a greater reduction in GP value than 5-N and 2,6-DH when Laurdan was excited by FRET. These results show that 1-N has the greatest effect in the lipidic domains near the gramicidin channel. On the other hand, excimer-monomer ratios of PDA obtained by FRET show that 1-N reduces the lateral mobility of acyl chains near the lipid-gramicidin interface when gramicidin concentration in the lipid bilayer increases. This effect is more noticeable than that obtained by direct irradiation of the probe in the presence of 5-N and 2,6-DH. On the other hand, the addition of the three alkanols in the presence of Gr produces a noticeable increase in the water permeability, particularly for 1-N. In this context, we propose a scheme that represents the effect of 1-nonanol on the water outflow in DPPC LUVs in the absence and in the presence of Gr.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Álcoois Graxos/química , Gramicidina/química , Bicamadas Lipídicas/química , Peptídeos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Transferência Ressonante de Energia de Fluorescência/métodos , Interações Hidrofóbicas e Hidrofílicas , Lauratos/químicaRESUMO
The bleaching of the pyrogallol red (PGR) dye mediated by superoxide anion radicals (O(2)(-)) generated from the xanthine/xanthine oxidase system (X/XO) was studied by UV-visible spectrophotometry. The absorption band (at 540 nm) of PGR quickly decreased in the presence of X/XO, implying an efficient reaction of O(2)(-) with PGR. The process was unaffected by catalase (CAT), but completely abolished by superoxide dismutase (SOD). A mechanism of the reaction involving the consumption of one PGR molecule by two O(2)(-) to generate one molecule of H(2)O(2) is proposed. PGR was used as a probe to estimate the rate of O(2)(-) generation in redox cycling reactions of a series of nitro compounds mediated by rat liver microsomes. The consumption of PGR induced by the redox cycling of nitrofurantoin was totally eliminated by the addition of SOD but unaffected by CAT. The initial rate of consumption of PGR mediated by the redox cycling of others nitro derivatives follows the order: furazolidindione > nitrofurantoin > nifurtimox > benznidazole > chloramphenicol. We concluded that PGR can be used as a probe to estimate the release of O(2)(-) from enzymatic systems or from the redox cycling of nitro compounds.
Assuntos
Nitrocompostos/metabolismo , Pirogalol/análogos & derivados , Superóxidos/química , Animais , Citocromos c/metabolismo , Etídio/análogos & derivados , Peróxido de Hidrogênio , Masculino , Microssomos Hepáticos/metabolismo , Oxirredução , Pirogalol/metabolismo , Ratos , Ratos Sprague-Dawley , Xantina/metabolismo , Xantina Oxidase/metabolismoRESUMO
Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg5² in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu², Phe¹5) did not modify the toxin's permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation.
Assuntos
Proteínas Citotóxicas Formadoras de Poros/química , Animais , Arginina/química , Arginina/genética , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Clonagem Molecular , Cisteína/química , Cisteína/genética , Mutagênese Sítio-Dirigida , Mutação , Compostos Orgânicos/química , Compostos Orgânicos/toxicidade , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Estrutura Terciária de Proteína , Anêmonas-do-Mar/metabolismo , Relação Estrutura-AtividadeRESUMO
A comparison of alizarin red (AR) and fluorescein (FL) as target molecules in oxygen radical absorbance capacity (ORAC)-like methods is reported. Galangin, apigenin, ferulic acid, and coumaric acid decreased AR initial consumption rate, whereas quercetin, kaempferol, luteolin, caffeic acid, and sinapic acid inhibited its consumption through an induction time, associated with a repair mechanism. On the other hand, all compounds protected FL with a clear induction time. AR was more selective and provides ORAC-AR values considerably smaller for compounds of low reactivity. The ORAC-AR value for luteolin was nearly 200 times that of coumaric acid. However, the ratio of ORAC-FL values for luteolin and coumaric acid was only 1.2. This different selectivity implies that AR provides ORAC values more related to reactivity than FL. ORAC-AR values of infusions were considerably smaller than the corresponding ORAC-FL values. These differences are interpreted in terms of the capacity of FL to generate induction times, irrespective of the reactivity of the additive. It is proposed that comparison of ORAC-AR and ORAC-FL values could afford a rough estimation of the average reactivity of the antioxidants titrated by the ORAC-FL methodology.
Assuntos
Antraquinonas/química , Antioxidantes/química , Fluoresceína/química , Corantes Fluorescentes/química , Espécies Reativas de Oxigênio/química , Espectrometria de Fluorescência/métodos , AbsorçãoRESUMO
The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.
Assuntos
Antioxidantes/metabolismo , Ácido Ascórbico/sangue , Ácido Ascórbico/urina , Pirogalol/análogos & derivados , Espécies Reativas de Oxigênio/sangue , Espécies Reativas de Oxigênio/urina , Glutationa , Humanos , Cinética , Pirogalol/sangue , Pirogalol/urinaRESUMO
DPPC incorporation into egg-PC unilamellar vesicles reduces their oxidation rate beyond that expected from the unsaturated lipid dilution. Addition of the unsaturated lipids produces changes in the physical properties of the inner parts of the lipid bilayer, as sensed by fluorescence anisotropy of DPH, and in the hydrophilic/hydrophobic region, as sensed by the generalized polarization of laurdan. DPPC (30 mol%) incorporation into egg-PC vesicles produces a decrease in alkyl chain mobility in the inner part of the bilayer, evaluated by the increase of DPH fluorescence anisotropy, and a rise of the generalized polarization value of laurdan in the bilayer interface. It also leads to a decrease in the rate of water efflux promoted by a hypertonic shock. Oxidation of PC LUVs, promoted by AAPH, as sensed by oxygen uptake and MDA formation, leads to qualitatively similar results than DPPC addition: rigidification at the inner part and the surface of the liposomes, and a lower rate of water permeation. It is suggested that these changes could contribute to the observed decrease in oxidation rate with conversion.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Peroxidação de Lipídeos , Fosfatidilcolinas/química , Anisotropia , Polarização de Fluorescência , Bicamadas Lipídicas , Lipídeos/química , Lipossomos/química , Fluidez de Membrana , Microscopia de Fluorescência/métodos , Oxigênio/metabolismo , Consumo de Oxigênio , Permeabilidade , Peróxidos/metabolismo , Água/químicaRESUMO
Sticholysins I and II (Sts I and II) are two potent cytolysins from the sea anemone Stichodactyla helianthus. These isoforms present 13 substitutions, with three non-conservative located at the N-terminus. St II is considerably more hemolytic than St I in human red blood cells, a result explained by the smaller number of negatively charged groups present at St II's N-terminus. In the present work, we have obtained a recombinant St I (rSt I), differing from the wild type in a single amino acid residue (E16Q). This pseudo-wild type is structurally similar to St I and shows a similar capacity to interact with and form pores in model membranes. This was assessed by the intrinsic fluorescence increase in the presence of liposomes, their adsorption to bilayers (measured by SPR), their concentration at the air-water interface, their interaction with lipid monolayers and their capacity to promote the release of carboxyfluorescein entrapped in liposomes. In spite of these similarities, rSt I presents a larger hemolytic activity in human red blood cells than St I, being intermediate in activity between Sts I and II. The results obtained in the present work emphasize that even the change of one single E by Q at the N-terminal segment may modify the toxin HA and show that this functional property is the most sensitive to subtle changes in the protein primary structure.
Assuntos
Proteínas Citotóxicas Formadoras de Poros/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , Eritrócitos/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Compostos Orgânicos/química , Compostos Orgânicos/isolamento & purificação , Compostos Orgânicos/metabolismo , Permeabilidade/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Ressonância de Plasmônio de Superfície , Tensão Superficial/efeitos dos fármacosRESUMO
Oxygen radicals absorbance capacities (ORAC) indexes are frequently employed to characterize the radical trapping capacity of pure compounds and their complex mixtures. A drawback of ORAC values obtained using phycoerythrin, fluorescein (FL) or c-phycocyanin as targets, makes it possible to conclude that for very reactive compounds they are much more related to stoichiometric factors than to the reactivity of the tested compound. In the present paper, we propose a simple methodology, based on the bleaching of Pyrogallol Red (PGR) absorbance that provides ORAC indexes that are almost exclusively determined by the reactivity of the tested compounds. This difference is due to the high reactivity of PGR and the high concentrations of this compound employed in the experiments.
Assuntos
Oxigênio/química , Peróxidos/química , Pirogalol/análogos & derivados , Espécies Reativas de Oxigênio/química , Radicais Livres/química , Estrutura Molecular , Pirogalol/química , Fatores de TempoRESUMO
The capacity of urocanic acid to interact with peroxyl radicals has been evaluated in several systems: oxidation in the presence of a free radical source (2,2'-azobis(2-amidinopropane; AAPH), protection of phycocyanin bleaching elicited by peroxyl radicals, and Cu(II)- and AAPH-promoted LDL oxidation. The results indicate that both isomers (cis and trans) are mild peroxyl radical scavengers. For example, trans-urocanic acid is nearly 400 times less efficient than Trolox in the protection of the peroxyl radical promoted bleaching of phycocyanin. Regarding the removal of urocanic acid by peroxyl radicals, nearly 100 muM trans-urocanic acid is required to trap half of the produced radicals under the employed conditions (10 mM AAPH, 37 degrees C). Competitive experiments show that the cis-isomer traps peroxyl radicals 30% less efficiently than the trans-isomer. Given the high concentrations that trans-urocanic acid reaches in skin, its capacity to trap peroxyl radicals could contribute to the protection of the tissue towards ROS-mediated processes. Furthermore, both isomers, and particularly the cis-isomer, protect LDL from Cu(II)-induced oxidation.
Assuntos
Amidinas/química , Peróxidos/química , Ácido Urocânico/farmacologia , Sulfonatos de Arila/farmacologia , Proteínas de Bactérias/metabolismo , Cromanos/farmacologia , Cobre/química , Radicais Livres , Lipoproteínas LDL/química , Modelos Químicos , Estresse Oxidativo , Oxigênio/metabolismo , Ficocianina/química , Espécies Reativas de Oxigênio , Spirulina , Ácido Urocânico/metabolismoRESUMO
Sticholysin II (St II) is a highly hemolytic cytolysin isolated from the sea anemone Stichodactyla heliantus. The toxin hemolytic action takes place through the formation of channels that provoke an electrolyte unbalance leading to osmotic shock. The lytic event must involve the exchange of electrolytes and the entrance of water, leading to red blood cell disruption. These processes can occur through St II pores and/or the endogenous red blood cells transporters. In order to evaluate the contribution of these channels to water, anion and cation transport, we have measured the hemolysis and K+ efflux rates in the presence of several specific inhibitors. The results obtained in the presence of Hg, an AQP1 blocker, indicate that water transport through these channels is not essential for the occurrence of the lytic process induced by St II. The data also support a partial role of K+ and anion transporters. In particular, they are compatible with a preferential K+ efflux though the K(+)/Cl- co-transport as a response to the promoted swelling. Furthermore, they suggest that chloride influx, a process that can regulate both K+ efflux and lysis, is partially mediated by the endogenous cell transporters, in particular, band-3 anion exchange system being relevant at early stages of the lytic process.
Assuntos
Venenos de Cnidários/toxicidade , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/toxicidade , Hemólise/efeitos dos fármacos , Canais Iônicos/metabolismo , Animais , Bário/metabolismo , Cálcio/metabolismo , Venenos de Cnidários/metabolismo , Eritrócitos/metabolismo , Proteínas Hemolisinas/metabolismo , Canais Iônicos/efeitos dos fármacos , Cloreto de Mercúrio/metabolismo , Potássio/metabolismo , Fatores de TempoRESUMO
A study has been made of the effect of urea upon the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase from Rhizopus arrhizus in AOT-heptane-water reverse micellar solutions at pH 7. The partition constants, K, of 2-NA between n-heptane and aqueous urea solutions in the absence of micelles were also determined. It was found that K decreases when the concentration of urea increases. In aqueous solution the rate of hydrolysis of 2-NA catalyzed by lipase is dependent on the concentration of urea (at a given 2-NA concentration). This result can be due to a decrease in the magnitude of the association of lipase with 2-NA and/or to changes in the reaction rate of the lipase-2-NA complex. The modifications of the enzymatic activities elicited by addition of urea show a lineal correlation with K, emphasizing the relevance of hydrophobic effects in the loss of activity. Nevertheless, the slope of the line is higher than one, suggesting that changes in the conformation of the enzyme would be also important. Addition of urea to the micellar solutions provokes a decrease of the enzyme activity. From the dependence of the reaction rate with AOT concentration, the partition constant of 2-NA between n-heptane and the micelles, K(p), was obtained. In the presence of 2 M urea a value of K(p)=0.33 M(-1) was derived. This value is lower than that measured in the absence of urea (Aguilar et al., Arch. Biochem. Biophys. 388 (2001) 231), indicating that incorporation of urea to the micellar interface produces a decrease of the association of 2-NA with the micelles. From a comparison of the results obtained in the micellar solution and in aqueous solution, it is concluded that the enzyme is more resistant to denaturation by urea in the micellar solution than in aqueous solution. Furthermore, at intermediate urea concentrations (2 M), the additive produces an increase in the Michaelis constant (K(M)) without a significant decrease (or even a small increase) in the catalytic rate constant (k(cat)).
Assuntos
Lipase/química , Micelas , Ureia/química , Catálise , Relação Dose-Resposta a Droga , Heptanos/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Microscopia de Fluorescência , Ácidos Naftalenoacéticos/química , Fatores de Tempo , Água/químicaRESUMO
The amount of alcohol required to produce a microemulsion in a quaternary water-in-oil system was evaluated for a series of alcohols and hydrocarbon solvents of different size or topology. It was observed that the amount of n-hexanol and n-decanol required was similar in all the solvents considered. On the other hand, considerably higher concentrations of the branched alcohols (2,4-dimethyl-3-pentanol and 3-ethyl-3-pentanol) were required to produce the microemulsion, irrespective of the solvent topology (n-hexane or 2,2,4-trimethylpentane). From an analysis of the change in the analytical alcohol concentration with the surfactant concentration the amounts of alcohol present at the microaggregates' surface at the point of microemulsion formation were obtained. It is concluded that the high amounts of branched alcohols required are due to both less efficient incorporation at the interface and the larger number of alcohol molecules per surfactant required to stabilize the microemulsion.
RESUMO
Heterogeneous enzymatic assays (HEA), where an enzyme in solution acts upon an immobilized substrate, are been increasingly used. Given their high throughput and versatility they hold great potential for developing massive enzyme inhibitor screening. However, current HEA lack, in general, rigorous quantitative use. This is in part due to technical problems as a multiplicity of suboptimal substrate populations achieved with traditional immobilization techniques but, more importantly, is due to a poor understanding of the particular kinetic behavior of these systems. This paper addresses the kinetic features of HEA that arise from the very low amount of solid-phase substrate and the resulting inalterability of the free enzyme concentration during the assay, which classify HEA as enzyme quasi-saturable systems (EQSS). We assessed the optimal enzyme concentration working range and time of reaction. We also considered certain attributes of HEA for evaluating isosteric inhibitors. These studies were done on the basis of a simplified model for the kinetics of EQSS and a formal splitting of the functional factor of the analytical sensitivity of an enzymatic assay into [E(o)]/K(m)-dependent and temporal components.
Assuntos
Enzimas/análise , Enzimas/química , Técnicas Imunoenzimáticas/métodos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Enzimas/efeitos dos fármacos , Cinética , Sensibilidade e EspecificidadeRESUMO
Sticholysin II (St II) a potent cytolysin from the sea anemone Stichodactyla helianthus was obtained by recombinant procedures exhibiting six histidine residues in its N-terminus (St IIn6H). The functional comparison between St II and St IIn6H showed a lesser pore-forming ability for the recombinant than for the native in human or rat red blood cells (RBC) and in large unilamellar vesicles (LUV) of different phospholipid composition. However, binding of St IIn6H to small unilamellar vesicles (SUV) was higher with regard to St II. The explanation to the different permeabilizing capacity of both protein variants is not clear, but a different anchoring of St IIn6H to the lipid bilayer could delay the organization of the competent pore into membrane.
Assuntos
Membrana Celular/metabolismo , Venenos de Cnidários/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/fisiologia , Hemólise/fisiologia , Substituição de Aminoácidos , Animais , Permeabilidade da Membrana Celular/fisiologia , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Proteínas Hemolisinas/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Concentração Osmolar , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anêmonas-do-MarRESUMO
In the absence of redox-active transition metal ions, the removal of Tempol by Trolox occurs by a simple bimolecular reaction that, most probably, involves a hydrogen transfer from phenol to nitroxide. The specific rate constant of the process is small (0.1 M(-1) s(-1)). Metals can catalyze the process, as evidenced by the decrease in rate observed in the presence of diethylenetriaminepentaacetic acid (DTPA). Furthermore, addition of Fe(II) (20 microM ferrous sulfate and 40 microM EDTA) produces a noticeable increase in the rate of Tempol consumption.
Assuntos
Antioxidantes/farmacologia , Cromanos/química , Óxidos N-Cíclicos/farmacologia , Fenol/química , Cromatografia Líquida de Alta Pressão , Óxidos N-Cíclicos/química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/química , Hidrogênio/química , Cinética , Modelos Químicos , Oxigênio/metabolismo , Ácido Pentético/farmacologia , Marcadores de Spin , Fatores de TempoRESUMO
Gramicidin incorporation to DPPC or lecithin-PC large unilamellar vesicles (LUVs) leads to pore formation that, under hyper-osmotic conditions, produces a noticeable increase in the rate of trans-membrane water flow. This pore formation is more efficient in the more fluid lecithin-PC LUVs. Exposure of these vesicles to peroxyl radicals generated in the aerobic thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH), changes the physical properties of the bilayer (as sensed employing fluorescent probes), modifies gramicidin molecules (as sensed by the decrease in Trp fluorescence) and notably reduces the transbilayer rate of water outflow. In order to evaluate if this reduced water-transport capacity is due to changes in the membrane due to lipid-peroxidation and/or direct damage to gramicidin channels, results obtained in the oxidable vesicles (lecithin-PC) were compared to those obtained in DPPC vesicles. The data obtained show that most of the water transport efficiency loss can be ascribed to a direct disruption of gramicidin channels by AAPH derived peroxyl radicals.