RESUMO
Steady state fluorescence measurements, due to their relative simplicity and fast and easy implementation, are one of the most employed techniques for evaluating the non-covalent binding of small molecules to proteins. In the present review we discuss the main characteristics of solute binding and the experimental procedures that can be employed for evaluating both, the efficiency of the process and the number of binding sites. It is also discussed the possibility of determining the distance between endogenous fluorophores and non-covalently bound solutes. Albumins (human serum albumin and bovine serum albumin) are considered as model proteins due to their relevance as solute carriers, the extensive available data comprising binding of a large number of solutes, and the reduced number of intrinsic fluorophores which simplifies the data treatment. It is shown that, in spite of the apparent simplicity of the systems, extreme care must be exercised in data treatment and interpretation to avoid misleading results. This applies to the evaluation of binding constants, number of binding sites, and average distance between intrinsic fluorophores and non-covalently bound solutes associated to the proteins.
Assuntos
Albuminas/metabolismo , Animais , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Ligação Proteica , Soluções/químicaRESUMO
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
The effect of sodium dodecyl sulfate (SDS) upon the conformation and hemolytic activity of St I and St II strongly depends on its concentration. At relatively low surfactant concentrations (ca. 0.5-5mM range) the surfactant leads to the formation of aggregates, as suggested by the turbidity observed even at relatively low (micromolar range) protein concentrations. In this surfactant range, the proteins show an increase in intrinsic fluorescence intensity and reduced quenching by acrylamide, with an almost total loss of its hemolytic activity. At higher surfactant concentrations the protein adducts disaggregates. This produces a decrease in fluorescence intensity, increase in quenching efficiency by acrylamide, loss of the native tertiary conformation (as reported by the near UV-CD spectra), and increase in alpha-helix content (as evidenced by the far UV-CD spectra). However, and in spite of these substantial changes, the toxins partially recover their hemolytic activity. The reasons for this recovering of the activity at high surfactant concentrations is discussed.
Assuntos
Venenos de Cnidários/farmacologia , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Animais , Dicroísmo Circular , Venenos de Cnidários/química , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/química , Humanos , Compostos Orgânicos , Conformação Proteica/efeitos dos fármacos , Anêmonas-do-Mar , Dodecilsulfato de Sódio/administração & dosagem , Espectrometria de Fluorescência , Tensoativos/administração & dosagemRESUMO
N-hexadecyl-N-N'-dimethyl-3-ammonio-1-propane-sulfonate (BPS) is a zwitterionic surfactant that readily binds to sticholysins I and II, two sea toxins isolated from Stichodactyla helianthus. The binding constants, evaluated from changes in fluorescence intensities elicited by the surfactant, are approximately 0.5-0.7 microM(-1). The binding of the surfactant changes the conformation of the tertiary protein, without significant changes in its secondary structure, as reported from far-ultraviolet circular dichroism spectra. The changes elicited by HPS lead to loss of the native conformation (as reported from near-ultraviolet circular dichroism spectra) and to a shift of the intrinsic protein fluorescence toward longer wavelengths, an increase in fluorescence intensities and lifetimes, and a faster quenching by acrylamide. All these changes are indicative of a more expanded tertiary conformation. Despite this, the toxins fully retain their hemolytic activities, indicating that spectroscopic changes can be poor predictors of toxin activity.
Assuntos
Cnidários/química , Hemólise/efeitos dos fármacos , Tensoativos/farmacologia , Toxinas Biológicas/farmacologia , Animais , Dicroísmo Circular , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Tensoativos/metabolismo , Toxinas Biológicas/química , Toxinas Biológicas/isolamento & purificaçãoRESUMO
In a previous work [Chem. Phys. Lipids 2000 104, 49], we have derived the following rate law for the oxidation of lipids in compartmentalized systems: R(T)=(k(1)/k(t))(0.5) k(p) [In](0.5) c(0.5) [LH], where, R(T) is the total rate of oxidation, k(1) is the rate constant for the production of free radicals, k(t) and k(p) are the intra-particle rate constants for the termination and propagation sets, respectively, [In] is the concentration of a water-soluble initiator, c is the concentration of particles, and [LH] is the intra-particle concentration of oxidable lipid. In the present work, we have investigated on the applicability of the proposed kinetic rate law for a system where it takes place the oxidation of a reactive lipid incorporated into an inert matrix. With this purpose, we have measured the rate of oxidation of linoleic acid incorporated into dipalmitoylphosphatidylcholine vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride as a function of the initiator, particles, and intra-particle LH concentrations. The experimentally determined kinetic orders obtained were 0.54+/-0.02, 0.48+/-0.05 and 0.83+/-0.04 for the dependence of the oxidation rate with initiator, particles, and LH intra-particle concentrations, respectively, in agreement with those theoretically predicted. The lower value obtained for the kinetic order in LH is attributed to a change in k(t) with the increase in oxidable lipid intra-particle concentration. The main point to be emphazised from the results here obtained is that the kinetic rate law for the oxidation of lipids in compartmentalized systems can be significantly different than that observed when to the oxidation takes place in homogeneous solution.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , 2-Naftilamina/análogos & derivados , Amidinas/química , Ácido Linoleico/química , Peroxidação de Lipídeos , Lipossomos/química , Oxidantes/química , Corantes Fluorescentes , Cinética , Lauratos , Modelos Biológicos , Espectrometria de Fluorescência/métodos , TermodinâmicaRESUMO
The preparation and the solvatochromic behavior of two dyes, obtained by condensation of N,N'-dimethylbarbituric acid with dimethylaminobenzaldehyde and with 4,4'-bis(N,N-dimethylamino)benzophenone (Michler's ketone) are described. The latter dye is rather sensitive to the polarity of the medium, and in particular, to the hydrogen-bond-donor ability of protic solvents. The solvatochromism of both compounds is discussed in terms of the pi* and E(T)(30) solvent polarity scales and their differences in behavior interpreted with the aid of semiempirical calculations.
Assuntos
Barbitúricos/química , Benzofenonas/química , Corantes Fluorescentes/química , Pirimidinas/química , Pirimidinonas/química , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Solventes/química , Espectrofotometria Ultravioleta/métodosRESUMO
A simple method useful for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes entrapped in reverse micelles is proposed. The method is applied to the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/buffer/heptane reverse micellar solutions. In the presence of micelles, the relationship between the initial reaction rate and the analytical concentration of 2-NA was dependent on AOT concentration at a constant W ([water]/[AOT]) value. The dependence of the initial reaction rate profiles with [AOT] was analyzed according with the method proposed to obtain the partition constant of 2-NA between the micelles and the external solvent, Kp. A value of Kp = 2.7 L mol(-1) was obtained irrespective of the water content of the micelles (W from 5 to 20). The catalytic rate constant kcat in the micellar solutions was independent of [AOT] but slightly decreased with an increase in W from 2 x 10(-6) mol g(-1) s(-1) at W = 5 to 1.2 x 10(-6) mol g(-1) s(-1) at W = 20. The apparent Michaelis constant determined in terms of the analytical concentration of 2-NA increased with [AOT] at a given W and moderately decreased with W at a fixed [AOT]. The increase with [AOT] is accounted for by considering the partitioning of the substrate. After correction for the partitioning of 2-NA values of (Km)corr were obtained as 3.9 x 10(-3) mol L(-1) (W = 5), 4.6 x 10(-3) mol L(-1) (W = 10), 2.3 x 10(-3) mol L(-1) (W = 15), and 1.7 x 10(-3) mol L(-1) (W = 20). The rate parameters in the aqueous phase in the absence of micelles, were obtained as (kcat)aq = 7.9 x 10(-6) mol g(-1) s(-1) and (Km)aq = 2.5 x 10(-3) mol L(-1). In order to compare the efficiency of the enzyme in the micellar solution with that in aqueous phase, the values of (Km)corr were in turn corrected to take into account differences in the substrate activity, obtaining so a set of (Km)*corr values. The efficiency of the enzyme in the micellar solution, defined as the ratio, kcat/(Km)*corr, was found to be higher than in the aqueous phase, even at high water contents (W = 20). This higher efficiency is due to a significant decrease in (Km)*corr values.
Assuntos
Lipase/metabolismo , Ácidos Naftalenoacéticos/metabolismo , Rhizopus/enzimologia , Soluções Tampão , Catálise , Ácido Dioctil Sulfossuccínico/química , Heptanos/química , Hidrólise , Cinética , Lipase/química , Micelas , Especificidade por Substrato , Água/químicaRESUMO
Kinetic rate laws arising from theoretical expectations for the oxidation of lipids initiated by water-soluble free radicals in compartmentalized systems under different experimental conditions are deduced. In particular, the predictions for the kinetic reaction orders in: (a) intra-particle oxidizable compound concentration (at fixed number of particles and particle size), alpha; (b) number of particles or analytical lipid concentration (at fixed intra-particle concentration and particle size), beta and (c) initiator, gamma, are obtained. The reaction orders beta and gamma are determined by the fraction of initiator derived radicals captured by the particles (f) and the mean number of chain carrying radicals per particle (
Assuntos
Peroxidação de Lipídeos , Amidinas , Radicais Livres , Técnicas In Vitro , Cinética , Lipossomos , Tamanho da Partícula , Fosfatidilcolinas/química , Solubilidade , ÁguaRESUMO
A new neoglycolipid (AgH-1) bearing carbohydrate units that mimics the antigenic determinant of the O-blood group was synthesized and the effect of its incorporation in dipalmitoylphosphatidylcholine (DPPC): cholesterol liposomes was evaluated. The results obtained show that AgH-1 is readily incorporated into DPPC:cholesterol liposomes. The conditions leading to the optimal incorporation are the result of a compromise between incorporation efficiency and incorporation extent. The presence of AgH-1 produces liposomes of smaller size, with only small changes in the properties of the bilayer. However, the data obtained employing diphenylhexatriene and laurodan as fluorescence probes and merocyanine 540 as optical probe suggest that AgH-1 incorporation leads to a small rigidization of the liposomes at temperatures lower than ca. 42 degrees C.
Assuntos
Glicolipídeos/química , Glicolipídeos/farmacologia , Lipossomos/química , Antígenos de Grupos Sanguíneos/imunologia , Sequência de Carboidratos , Colesterol/metabolismo , Difenilexatrieno/metabolismo , Membrana Eritrocítica/química , Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Cinética , Bicamadas Lipídicas/química , Lipossomos/metabolismo , Dados de Sequência Molecular , Naftalenos/metabolismo , Tamanho da Partícula , Fosfatidilcolinas/metabolismo , Pirimidinonas/metabolismo , TemperaturaRESUMO
Osmotic shrinkage changes the surface properties of dipalmitoylphosphatidylcholine large unilamellar vesicles depending on the phase state of the bilayer. In the gel state, shrinkage produces an increase in the adsorption of hydrophobic dyes, such as Merocyanine 540 (MC540) monomers, toluidine and anilinonaphthalene sulfonic acid (TNS, ANS). In the fluid state, shrinkage does not affect the bilayer surface when gradients between the inner and the outer compartments below 0.2-0.25 M NaCl (higher concentration outside) are applied. Larger differences in concentrations produce an increase in packing as inferred from the desorption of the MC monomers. Kinetic experiments show that the surface changes correlate with the volume decrease produced by the water extrusion from the vesicle interior. It is interpreted that the decrease of water content compels the vesicles to a state in which defects at the membrane surface are likely to occur when the bilayer is in the gel state.
Assuntos
Lipossomos/metabolismo , Osmose/fisiologia , Propriedades de Superfície , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Corantes/metabolismo , Difenilexatrieno/metabolismo , Fluoresceínas/metabolismo , Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Géis/metabolismo , Cinética , Bicamadas Lipídicas/metabolismo , Nefelometria e Turbidimetria , Tamanho da Partícula , Pirimidinonas/metabolismo , Cloreto de Sódio/farmacologia , Espectrofotometria , Temperatura , Água/metabolismoRESUMO
The partition constants (K(p)) of iodine between sodium dodecylsulfate micelles and the external media have been measured in the presence of water-soluble (sodium sulfate) and micelle-soluble (n-hexane and n-hexanol) additives. A spectrophotometric method based in the analysis of the alterations provoked by the presence of the surfactant plus the additives upon the reversible reaction taking place between iodine and iodide to give tri-iodide ions was employed (Abuin, E., and Lissi, E., J. Chem. Ed. 69, 340 (1992)). The effect of the additives upon the n-octanol/water partitioning (K(o/w)) of iodine was also determined. The results obtained for K(p) and its relationship with the K(o/w) values are indicative of a preferential localization of iodine at the surface of the micelles.
RESUMO
The effects of pH and ionic micelles on the rates of product formation following irradiation of riboflavin in the presence of tryptophan were investigated by absorption and fluorescence spectroscopy. Under anaerobic conditions, formation of riboflavin-tryptophan adducts was inhibited in acid solutions and by the addition of anionic (sodium dodecylsulphate) and cationic (cetyltrimethylammonium bromide) micelles. The oxidation of tryptophan photoinduced by riboflavin was considerable faster in basic solutions.