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The behavior of 4-aminophthalimide (4-AP), a common molecular probe utilized in solvation dynamics experiments, was revisited in polar aprotic and protic solvents using absorption, steady-state, and timeresolved fluorescence (TRES) techniques. Also, the deuterium isotope effect was investigated using D2O as solvent. The absorption spectra of 4- AP consist of two absorption bands with maxima around 300 nm (B2 band) and 370 nm (B1 band) depending on the environment, while the emission feature consists of a single band. In all the solvents investigated (excluding water), the 4-AP photophysics is similar and the emission spectra are independent of the excitation wavelength used. In water the behavior is unique and the emission spectra maximum is different depending on the excitation wavelength used. The emission maximum is 561.7 nm using the excitation wavelength that correspond to the B2 absorption band maximum (λexcB2 = 303.4 nm) but is 545.7 nm when the excitation wavelength that correspond to the B1 absorption maximum (λexcB1 = 370.0 nm) is used. Moreover, while the fluorescence decays of 4-AP in water exhibit no emission wavelength dependence at λexcB2, the situation is quite different when λexcB1 is used. In this case, we found a time-dependent emission spectrum that shifts to the blue with time. Our results show that the solvent-mediated proton transfer process displays a fundamental role in the 4-AP emission profile and for the first time a mechanism was proposed that fully explains the 4-AP behavior in every solvent including water. The deuterium isotope effect confirms the assumption because the proton-transfer process is dramatically retarded in this solvent. Consequently, we were able to elucidate not only why in water the emission spectra depend on the excitation wavelength but also why the time-dependent emission spectra shift to the blue with time. Thus, our work reveals the importance that the medium has on the behavior of a widespread dye used as chromophore. This is significant since the use of chromophores without understanding its chemistry can induce artifacts into the interpretation of solvation dynamics in heterogeneous environments, in particular, those provided by aqueous biological systems. Fil: Durantini, Andres Matías. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Falcone, Ruben Dario. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Anunziata, Jorge Daniel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Abuin, Elsa B.. Universidad de Santiago de Chile; Chile Fil: Lissi, Eduardo A.. Universidad de Santiago de Chile; Chile Fil: Chessa, Juana Josefa. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The effect of human serum albumin (HSA) addition on the rate of hydrolysis of the synthetic substrate 4-methylumbelliferyl-β-D-N-N'-N″ triacetylchitotrioside ((NAG)(3)-MUF) catalyzed by hen egg white lysozyme has been measured in aqueous solution (citrate buffer 50 mM pH = 5.2 at 37 °C). The presence of HSA leads to a decrease in the rate of the process. The reaction follows a Michaelis-Menten mechanism under all the conditions employed. The catalytic rate constant decreases tenfold when the albumin concentration increases, while the Michaelis constant remains almost constant in the albumin concentration range employed. Ultracentrifugation experiments indicate that the main origin of the observed variation in the kinetic behavior is related to the existence of an HSA-lysozyme interaction. Interestingly, the dependence of the catalytic rate constant with albumin concentration parallels the decrease of the free enzyme concentration. We interpret these results in terms of the presence in the system of two enzyme populations; namely, the HSA associated enzyme which does not react and the free enzyme reacting as in the absence of albumin. Other factors such as association of the substrate to albumin or macromolecular crowding effects due to the presence of albumin are discarded. Theoretical modeling of the structure of the HSA-lysozyme complex shows that the Glu35 and Asp52 residues located in the active site of lysozyme are oriented toward the HSA surface. This conformation will inactivate lysozyme molecules bound to HSA.

The rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by alpha-chymotrypsin (alpha-CT) has been measured in aqueous solutions of dodecyltrimethylammonium bromide (DTAB) at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction follows a Michaelis-Menten mechanism. The presence of the surfactant leads to superactivity below and above the critical micelle concentration (CMC), with a maximum reaction rate taking place near the CMC when the results are treated in terms of the analytical concentration of the substrate. A similar behavior was observed by working with the enzyme partially deactivated in the presence of 4 M urea. After correction to take into account the partitioning of the substrate between the micelles and the external media, the activity of the enzyme tends to remain almost constant above the corresponding CMCs. This results from a compensation of a decrease in the catalytic constant (k(cat)) and a decrease in the Michaelis constant (K(M)). The behavior of alpha-CT in the hydrolysis of GPNA in DTAB solutions is at variance with that previously reported for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant (E. Abuin, E. Lissi, R. Duarte, Langmuir 19 (2003) 5374). An explanation of the different effects of the surfactant on the behavior of the enzyme with both substrates is advanced, taking into account the complexity of the mechanism of the alpha-CT-mediated reaction, more specifically, in terms of different rate-limiting steps for the formation of the measured products.

Fil: Durantini, Andres Matías. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Falcone, Ruben Dario. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Anunziata, Jorge Daniel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Chessa, Juana Josefa. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Abuin, Elsa B.. Universidad de Santiago de Chile; Chile Fil: Lissi, Eduardo A.. Universidad de Santiago de Chile; Chile Fil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Alkyl pyridinium ions readily quench the fluorescence arising from Trp groups incorporated to a dipeptide (Ala–Trp) or protein (BSA). The quenching of the dipeptide fluorescence takes place with a Stern–Volmer constant of 730 M −1 in aqueous solution, irrespective of the length of the alkyl chain (octyl, dodecyl or hexadecyl). On the other hand, the quenching of BSA fluorescence strongly depends upon the length of the alkyl chain. Octyl pyridinium ion presents a normal behavior, with K SV values of 310 and 280 M −1 in aqueous solution and in presence of 8 M urea, respectively. These values are independent of the protein concentration. Dodecyl and hexadecyl pyridinium ions are considerably more efficient as quenchers. Stern–Volmer plots show a strong upward curvature, the quenching efficiency decreases when the protein concentration increases, and is considerably reduced in presence of salt (phosphate) or urea. These results are explained in terms of a static quenching between the adsorbed alkyl pyridinium and the excited Trp groups. From the dependence of the quenching efficiency with the protein concentration it is obtained the alkyl pyridinium/BSA association constant. The values obtained indicate that the binding is dominated by hydrophobic (dependence with the alkyl chain length and urea concentration) effects and, in minor degree, by electrostatic interactions.

A methodology is proposed to investigate the origin of the autoacceleration frequently observed when the oxidation of lipids takes place in compartmentalized systems. The method is based on the evaluation of post-irradiation effects and time profiles of oxygen consumption when the irradiation is re-initiated after a dark period. The proposal is experimentally tested in the oxidation of egg-yolk phosphatidylcholine (PC) vesicles initiated by decomposition of a water soluble free radical source. Three sceneries and the expected time profiles of oxygen consumption are proposed to explain the origin of the autoacceleration effect: (i) initial inhibiton by remainig low levels of (unknown) antioxidants; (ii) autoacceleration due to contribution of peroxidized products to the initiation rate; and (iii) a lag time due to the long reactions times required to reach the steady-state condition when the oxidation takes place in compartmentalized systems. The results obtained are consistent with case (i). The conclusion reached is considered to be relevant in the context that it allows differentiation between spurious effects [case (i)] and intrinsic effects [cases (ii) and (iii)]. This can contribute to a better understanding of the factors involved in the interpretation of the kinetic and mechanistic aspects of lipid peroxidation in compartmentalized systems.

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 π * and E T (30) solvent polarity scales and their differences in behavior interpreted with the aid of semiempirical calculations.

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.

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 (n) when the system reaches the steady state condition. Predicted orders in initiator range from 0 (n= 0.5) to 0.5 (f--1;n1), while the order in number of particles ranges between 0.5 (f--1;n1) and 1. These predictions are tested by measuring the kinetic law for the oxidation of SUV's egg yolk phosphatidylcholine vesicles initiated by the thermal decomposition of ABAP. The results indicate that, under the conditions employed, beta = 0.68 +/- 0.05 and gamma = 0.46 +/- 0.04. These values are close to those expected for a system in whichn1 and the efficiency of capture is relatively high. This last condition is confirmed by estimating the efficiency of capture from a comparison of induction times elicited by similar concentrations of Trolox and alpha-tocopherol.

The partitioning of laurdan and prodan between the external solvent and the microaggregates in AOT/heptane/water water-in-oil microemulsions has been measured. We have also evaluated the dependence of the fluorescence spectra (as determined by the generalized polarization, GP) with the water content of the microemulsion and the characteristics of the emission and excitation GP spectra in order to assess the properties of the average microenvironments sensed by the microaggregate-bound probes. Partition constants (K, molar concentration units) were evaluated from the change in fluorescence intensity (at a fixed probe concentration) with the AOT concentration (at fixed W = (water)/(AOT) values). For W values ≥10, a value K = 1.0 ± 0.2 M-1 was obatined, both for laurdan and prodan. This result indicates that neither the length of the probe hydrophobic tail nor the size of the water pool significantly modifies the affinity of the probe for the microaggregates. Even lower values of K were obtained at lower W v...

Reverse micelles, or water-in-oil microemulsions, incorporate a variety of solutes. The distribution of the solute between the surrounding solvent and the micelles can be described in terms of a partition constant based on a two pseudophases model that can be expanded to three pseudophases in micelles with high water to surfactant mole ratios. In these systems, two partition constants can be defined, one between the external solvent and the micellar interface, and other between the external solvent and the inner water pool. The present review describes the methodologies that can be employed to obtain distribution constants, and discuss literature data obtained in these systems. The localization of solutes associated to the micelles, as well as the effects promoted by solute incorporation on micellar properties are also discussed.

The binding of sodium dodecylsulfate (SDS) to poly ( N -isopropylacrylamide), (PNIPAM) microgels at different temperatures (below and above the transition temperature from swollen to shrunken particles, T s ) has been investigated by fluorescence probe methods complemented with surface tension and dynamic light scattering measurements. Pyrene and 1-anilinonaphthalene-8-sulfonate (ANS) were employed as fluorescence probes. At 21°C, the association of the surfactant with the particles occurs at a critical aggregation concentration (cac). Values obtained were (cac)=1 mM form surface tension data and (cac)≅0.75 mM from the dependence of the fine structure of pyrene fluorescence spectra with SDS addition. The binding isotherm derived from surface tension data at 21°C is highly cooperative, implying that the binding takes place with formation of surfactant aggregates. At 42°C (a temperature above T s ), the (cac) shifts to 1.7 mM indicating that the interaction of SDS with shrunken PNIPAM microgels is weaker than with the swollen species. The microenvironment sensed by pyrene probe in both, the SDS aggregates formed onto PNIPAM swollen (21°C) and shrunken (42°C) species is less polar than that corresponding to SDS micelles in pure water. The T s of the particles and the effect of SDS on it can be determined from the temperature dependence of ANS fluorescence intensity or emission band position. At temperatures higher than T s , coulombic repulsion effects (probably due to the higher charge density of the collapsed particles) are evidenced by the fluorescence behaviour of both probes employed.

Vesicles of dioctadecyltrimethylammonium chloride were prepared by sonication, membrane extrusion and dichloromethane injection. The diameter of the vesicles were determined using quasi-elastic light scattering and freeze fracture electron microscopy. The incorporation of water soluble probes in the vesicles allowed the determination of a molar volume of 6 to 8 L/mole for the CH2Cl2 injected vesicles and 1.0 L/mole for the membrane extruded vesicles, in very good agreement with the data calculated from the vesicle diameters. The permeability of the injected CH2Cl2 vesicles is dependent on temperature and probe structure. Membrane extruded vesicles are more permeable than injected vesicles, both below and above the phase transition temperature.

Fluorescence quenching techniques complemented with optical rotation and viscosity measurements were employed to study the effects of urea and sodium dodecylsulfate on the formation of hydrophobic microdomains and its relationship to the extent of helical structuration of gelatin chains in aqueous solution. The effects were examined for both additives acting independently of each other as well as for their joint action. Addition of urea (0.2–2 M ) to a 0.5% (w/v) gelatin solution decreases the fraction of the fluorescence of pyrene protected against quenching by acrylamide in a fashion directly related to the extent of triple helix structuration of the polypeptide chains. The decrease in the amount of structured microdomains elicited by urea addition is accompanied by a substantial decrease in the viscosity of the gelatin solution. Upon addition of 1 m M SDS to the gelatin solution at 0.5% (w/v), the fluorescence of pyrene is almost completely protected against quenching by acrylamide, the triple helix content slightly decreases, and the viscosity of the solution increases. The joint incorporation of both additives produces an increase in the viscosity of the solution when SDS is added to a gelatin solution containing urea but, at any SDS concentration, the presence of urea decreases the viscosity of the solution. Similarly, addition of SDS to a highly disorganized gelatin solution in the presence of urea increases the fraction of triple helix, but urea added to the gelatin solution in the presence of SDS decreases it. The effects observed are interpreted in terms of the formation of different types of microdomains when urea or SDS are added independently as well as when they are simultaneously present in the solution.

The partition constants (Kp) 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 andn-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 then-octanol/water partitioning (Ko/w) of iodine was also determined. The results obtained forKpand its relationship with theKo/wvalues are indicative of a preferential localization of iodine at the surface of the micelles.

Results obtained on the effect of addition of dodecyltrimethylammonium bromide (DTAB) upon the α-chymotrypsin (α-CT) catalyzed hydrolysis of 2-naphthyl acetate (2-NA) under steady state conditions for the acyl–enzyme intermediate are compared with those previously obtained in the transient (pre-steady state or “burst”) phase. It is found that, while in the transient phase there is no effect of DTAB addition on the kinetic parameters at concentrations below the critical micelle concentration (CMC) of the surfactant, super-activity is observed when the acyl–enzyme intermediate reaches the steady state condition. This difference implies that the surfactant does not modify either the formation or the decomposition of the enzyme–substrate complex (transient phase) but notably increases the rate of disruption of the acyl–enzyme intermediate.

We have investigated the effect of the incorporation of a linear (1-octanol) and a branched (2,6-dimethyl-4-heptanol) alkanol into large unilamellar vesicles of dipalmitoylphosphatidylcholine in the gel state upon several properties of the bilayer. The effect was evaluated by measurements of the rate of water outflow following a hypertonic osmotic shock, generalized polarization of 6-dodecanoyl-2-dimethyl)aminonaphthalene (Laurdan), absorption spectra of merocyanine 540 (MC), fluorescence anisotropies of 2-anthroyloxystearic acid (2-AS), 12-anthroyloxystearic acid (12-AS) and diphenylhexatriene (DPH), the lifetimes of pyrene (Py) and pyrenedodecanoic acid (PyD), and the pseudo-first order rate constant of the fluorescence quenching of Py and PyD by oxygen, kox, as a function of the intravesicular alkanol concentration. The data obtained indicate that the degree of incorporation, and the effect of a given intravesicular concentration of the additive, are extremely dependent on the molecular shape of the alkanol. In particular, the branched alkanol is less soluble and causes greater disorganization of the central parts of the bilayer than the linear one. The incorporation of both additives at low alkanol/lipid ratios produces a notable increase in the rate of water outflow following a hypertonic shock, the effect being more noticeable at low concentrations of the branched alcohol. Both alkanols produce, at least at low concentrations, an increase in the value of 2-AS fluorescence anisotropy, suggesting a more compact organization of the interface. In this region, MC is expelled from the vesicle. At higher 1-octanol concentrations, the fluorescence anisotropy of 2-AS decreases and MC incorporation increases, indicating a biphasic behaviour for the linear alcohol. Significant differences between both alkanols are also observed with respect to their effect upon the values of kox for PyD, a probe that can be considered to be localized in the inner parts of the bilayer.

The decomposition of hydrogen peroxide catalyzed by catalase entrapped in the pool of di-palmitoylphosphatidyl choline unilamellar liposomes has been studied. The rate of the process was evaluated by following the production of oxygen as a function of time. Under the experimental conditions employed the rate of oxygen production was controlled by the diffusion of hydrogen peroxide, allowing for the estimation of the diffusion coefficient of hydrogen peroxide across the liposome bilayer. The rate of diffusion across the bilayer increases with the temperature and the presence of fluidizers (n-nonanol), according with changes in the bilayer fluidity, as sensed by 1,6-diphenyl hexatriene (DPH) fluorescence anisotropy. A peculiar aspect of the data is the fast hydrogen peroxide diffusion observed at the bilayer phase transition temperature. This fast diffusion is associated to rafts fluctuations that take place in the partially melted bilayer. These fluctuations have no effect on the microviscosity sensed by DPH.

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.

To elucidate the role of fixed charge and chain rigidity on the assembly of amphiphilic compounds over a polymer substrate, we studied the interaction between sodium dodecyl sulfate (SDS) and crystalline or soluble collagen. Shrinkage temperatures and the equilibrium degree of swelling were measured for crosslinked tendons under isoelectric (pH=6.0) conditions or in acid (pH=2.5) solutions. Viscosities and solubilities were measured for soluble collagen to construct pseudo phase diagrams delimiting the field of stability of the helical, random coiled, and crystalline forms. Under isoelectric conditions, increasing concentrations of SDS (up to ≃0.1 M) cause a depression of transformation temperatures which is much larger than that observed with salts, with aliphatic alcohols, or with sodium methyl sulfate

3-(3-indoyl)-propyl methacrylate (IPM) was prepared and subsequently copolymerized with methylmethacrylate (MMA) in order to obtain a poly-(methyl methacrylate) containing fluorescent indolic groups (PMMI). 3-(3-indoyl)-propyl isobutyrate (IPI), was also synthetized as a model compound. Characterization of the polymer gave a molecular weight of 200,000 and a mean average number of 2.5 indolic units per chain. The model compound and the polymer showed the same absorption spectra, with a maximum at 280 nm. The emission spectra for both compounds were strongly dependent on the solvent, with a shift of the emission wavelengths towards the red in polar solvents. In a given solvent, both polymer and model emit at the same wavelength, the only exception for this was observed when 1-propanol was employed as solvent. This was attributed to a “chain effect” exerted by the strongly folded macromolecule, shifting the emission wavelength toward values corresponding to polarities lower than the bulk solution. In solvent mixtures, the maximum emission wavelength shifts can be considered as indicative of the probe environment, reflecting a sort of preferential adsorption of the component with the highest thermodynamic power onto the macromolecular chains. Finally, a number of quenching experiments were performed in pure solvents and solvent mixtures, showing that the deactivation process is diffusion-controlled. The lower values found for the polymer could indicate that the quencher finds a certain resistance to penetrate the polymer coil, which might be due to steric effects or a higher local viscosity within the macromolecular coil.

The effect of poly (ethyleneglycol) (PEG) addition on the rate of p-nitrophenyl acetate (PNPA) catalyzed by a- chymotrypsin has been measured in TRIS buffer at pH = 7.4. The presence of PEG (up to 30 % p/v) leads to an increase in the rate of the process. The effect of PEG is evident both, in the pre-steady state and steady state stages of the process. The reaction follows a Michaelis-Menten mechanism under all the conditions employed. The Michaelis constant slightly decreases and the catalytic rate constant slightly increases with PEG concentration over all the polymer concentration range employed (up to 30%). The decrease in the Michaelis constant suggests that the effect of the polymer is due to crowding.

The effect of human serum albumin (HSA) addition on the rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by α-chymotrypsin has been measured in phosphate buffer saline at pH = 7.4. The presence of HSA (up to 200 μM) leads to a decrease in the rate of the process. The reaction follows a Michaelis–Menten mechanism under all the conditions employed. To take into account the effect of substrate depletion due to its binding to albumin ultrafiltration experiments were carried out from which the binding of GPNA to HSA was derived. After correction of the kinetic data taking into account the binding of GPNA to HSA, the activity of the enzyme, and the derived Michaelis constant and catalytic rate constant tends to remain almost independent of the presence of albumin, indicating that the depletion of the substrate due to its binding to HSA is the main factor affecting the enzyme activity.

The number of binding sites for a given solute in a protein is a most relevant parameter. This number can be derived from fluorescence quenching data which provides the fraction of sites occupied at a given free solute concentration. Data are generally treated according to Scatchard´s or Ward´s equations. Lately, a double logarithmic plot of the data has been extensively used with this purpose. The present communication discus the validity of this procedure. It is concluded that this type of plot provides an evaluation of the stoichiometry (molecularity) of the binding process but not the number of equivalent binding sites per protein.

The association and photobehavior of Rose Bengal (RB) in the presence of dipalmitoylphosphatidyl choline (DPPC) small unilamellar liposomes is determined by the temperature. At temperatures above the main phase transition of the bilayer, the incorporation of the dye is ca. 2.5 times more efficient than that taking place when the bilayer is in the gel state. In both temperature ranges, adsorption isotherms show a noticeable anti-cooperativity that can be related to electrostatic repulsion between bound molecules. The photophysics and the photochemistry of the bound dye molecules also depend on the bilayer status. In particular, in the liquid crystalline state the surrounding of the dye is more polar and production of singlet oxygen is less efficient ( Φ ∼0.1). This reduced singlet oxygen production is partially due to a low triplet yield ( Φ T =0.35) and triplet self-quenching due to a high local RB concentration. In spite of these, tryptophan is efficiently photobleached when RB is associated to liposomes in the liquid crystalline state, probably due to a Type I mechanism favored by its high local concentration in the sensitized surroundings.

The solubility of water in water-in-oil microemulsions stabilized by cetyltrimethylammonium (CTA) has been investigated as a function of the nature of the surfactant counterion (bromide; CTAB/chloride; CTAC), the composition of the oil (chloroform and chloroform/heptane mixtures), the salinity of the droplets, and the nature of the salt employed to modify it. Changing the composition of the oil from neat chloroform to a 50% (v/v) mixture with heptane produces an increase of the water solubility in the microemulsions stabilized by CATB and decreases it in the CTAC microemulsions. Strong alterations of the solubility of water were found to occur upon mixing of the surfactants. Solubility increases abruptly on going from either CTAC- or CTAB-stabilized microemulsions to mixtures of both surfactants, reaching a maximum at CTAC mole fractions of 0.5 and 0.4 for solutions of the surfactants in chloroform and chloroform/heptane (3/1 v/v), respectively. Mixtures of CTAC and CATB in a solvent comprising 50% chloroform/heptane (v/v) show a different behavior. In this system, water solubility continuously decreases when the CTAC/CTAB ratio increases. Increasing the salinity of the droplets affects in an opposite fashion the solubility of water in the CTAC- and in the CTAB-stabilized microemulsions, irrespective of the nature of the salt employed. Solubility increases with salinity in the CTAC-stabilized microemulsions and decreases in the CTAB-stabilized microemulsions. Nevertheless, the magnitude of the effects observed is dependent on the nature of the salt employed to modify the salinity of the droplets. Results obtained from experiments in which equivalent salinities were reached by addition of different salts make it possible to differentiate salt effects from counterion-specific effects. The results obtained are explained in terms of the model of Shah and co-workers ( Langmuir 3, 1086 (1987); J. Colloid Interface Sci. 120, 320 and 330 (1987)) by considering that, due to the stronger binding of bromide than of chloride to the surfactant heads at the micellar interface, water solubility in solutions of CTAB or CTAC in chloroform is determined by different factors: the curvature of the surfactant film (CTAB) and the interaction between droplets (CTAC).

The rate of quenching of aromatic hydrocarbon fluorescence by olefins is extremely dependent upon the solvent, being particularly fast in aqueous solutions. The possibility that this solvent effect is partly due to solvophobic interactions and/or to stabilization of a charge transfer intermediate is discussed. From an analysis of results obtained in several polar solvents, as well as from the effect of temperature and the addition of solutes with “salting-in” and “salting-out” capacities, it is concluded that the data in aqueous solution are better interpreted in terms of the stabilization afforded to the intermediate stages of the process by a hydrophobic effect arising from the decrease in water—solute interfacial area along the reaction coordinate.

Competition between Tl+ and Na+ ions to bind with the surfaces of mixed micelles composed of dodecylsulfate and Brij 35 surfactants has been studied by a fluorescence-quenching technique. The quenching data are interpreted in terms of the pseudophase ion-exchange formalism. It is found that the competitive binding is well described by a unique K( Tl + Na + ) = 2.6 ± 0.2 selectivity coefficient irrespective of the micelle's composition within the range considered (mole fraction of the ionic component in the micelle between 1 and 0.5). The results further reinforce previous conclusions obtained in a cationic/nonionic micellar system [E. Abuin and E. Lissi, J. Colloid Interface Sci.143, 97 (1991)] by showing that selective binding of counterions to mixed micelles is almost totally related to the nature of the exchanging counterions and depends very little on the composition of the micelles.

A relevant question concerning the kinetics of reactions catalyzed by water-soluble enzymes in reverse micellar solutions is whether the efficiency of the enzyme is different from that in bulk aqueous solution. The comparison can be carried out only if the rates of the processes are compared under conditions of equal substrate activity. In the present work, it is proposed that this comparison can be carried out by employing the activity of the substrate in bulk water solution as a thermodynamic concentration scale. In order to carry out this comparison, the kinetic results obtained in the reverse micellar solution employing the analytical substrate concentration must be corrected by the solute distribution between the micellar pseudophase and the external solvent and by the partitioning of the substrate between the external solvent and an aqueous solution. The proposed methodology is applied to data previously reported for the oxidation of aliphatic alcohols catalyzed by alcohol dehydrogenase in a sodium ...

A study has been made on the properties of human serum albumin (HSA) binding sites and how they are modified by pre-oxidation of the protein with hypochlorite. The oxidation extent was assessed from changes in the protein intrinsic fluorescence and production of carbonyl groups. HSA retains its solute binding capacity even after exposure to relatively large amounts of hypochlorite (up to 40 oxidant molecules per protein). From an analysis of the binding isotherms of dansyl sarcosine (DS) and dansyl-1-sulfonamide (DNSA) to native and hypochlorite treated albumin it is concluded that pre-oxidation of the protein reduces the number of active sites without affecting the binding capacity of the remaining binding sites. From DS and DNSA fluorescence anisotropy, Laurdan anisotropy and generalized polarization measurements, it is concluded that both Sites I and II in the native protein provide very rigid environments to the bound probes. These characteristics of the sites remain even after extensive treatment with hypochlorite. This stubbornness of HSA could allow the protein to maintain its function along its in vivo lifetime.

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 considerably faster in basic solutions.

The effect of the identity of the counter-ion on the structural characteristics of dodecylsulphate micelles was investigated using techniques based on the interpretation of the IR and fluorescence spectroscopic behaviour of guest molecules. 6-Undecanone and pyrene were used as guests in the IR and fluorescence studies respectively. The counter-ions considered were lithium, sodium, tetramethylammonium and tetraethylammonium ions. The structural features of the micelles were revealed by monitoring parameters which allow for a qualitative analysis of the extent of exposure of the guest molecules to water. The spectroscopic behaviour of the probes was also examined in the micellar solutions in the presence of additives (n-hexanol and salts). The results obtained indicate that, of the counter-ions investigated, lithium promotes the formation of micelles with the roughest surfaces, followed in decreasing order by sodium, tetramethylammonium and tetraethylammonium ions. The micelles modified by incorporation of n-hexanol or addition of salt induce the displacement of the probes from the surface; the effect is greatest in lithium dodecylsulphate micelles.

The competition between bromide and chloride for the binding at the surface of mixed micelles composed of tetradecyltrimethylammonium and Brij 35 surfactants has been studied by fluorescence quenching techniques. The results obtained conform to the pseudophase ion-exchange formalism over a wide range of micelle compositions and allow for the determination of the selectivity coefficients. It was found that the competitive binding is well described by a uniqueK(Br/Cl) = 6.3 ± 1 selectivity coefficient, irrespective of the micelle's composition within the range considered (mole fraction of the ionic component in the micelle from 1 to 0.4). The results indicate that the factors involved in determining the selective binding are almost totally related to the nature of the exchanging counterions and very little dependent on the characteristics of the micelle surface and superficial charge density.

Small and large vesicles were prepared with dioctadecyldimethylammonium chloride and bromide. The hydrodynamic radius of the small vesicles ranges from 11 to 30 nm, while that of the large vesicles is ca. 140 nm. Both small and large vesicles increased the rate of alkaline hydrolysis and thiolysis (heptyl mercaptan, HM) of p-nitrophenyl octanoate (NPO). The rate/amphiphile patterns were different and the small vesicles were 2-5 fold more effective as catalysts. From the quantitative analysis of the kinetic results, using a pseudophase model with ion exchange, we concluded that the size dependence is due to differences in ion dissociation, substrate binding constants, and intrinsic reactivities

A study has been made of the effect of sodium dodecylsufate (SDS) addition on the oxidation of ethanol catalyzed by yeast alcohol dehydrogenase. Experiments were performed at pH = 8.1 and SDS concentrations employed were below and above the surfactant critical micelle concentration (CMC). The double reciprocal plots obtained in the absence and in the presence of the surfactant were compatible with a sequential bi-bi ordered mechanism. In the presence of the surfactant the initial reaction rates were consistently lower than in pure buffer at all the surfactant concentrations considered (0.5-50 mM). This effect is mainly due to an increase in the dissociation constant of beta-NAD(+) which reaches its maximum value (7,100 +/- 1,700 microM) at the CMC. Above the CMC the effect of the surfactant is mainly due to an increase in the Michaels constants of the alcohol, with values of 41 +/- 1 mM for 15 mM SDS and 50 +/- 1 mM for 50 mM SDS. The catalytic rate constant was found to be practically independent of the presence of the surfactant in the range of concentrations considered (up to 50 mM).

The effect of surfactants, both in water-in-oil microemulsions (hydrated reverse micelles) and aqueous solutions upon enzymatic processes is reviewed, with special emphasis on the effect of the surfactant upon the kinetic parameters of the process. Differences and similarities between processes taking place in aqueous and organic solvents are highlighted, and the main models currently employed to interpret the results are briefly discussed.

A study has been made on the interaction of cetylpyridium chloride (CPC) and dodecylpyridinium chloride (DPC) with human serum albumin (HSA) in aqueous solution (22 °C, pH = 7.0). The study was performed using techniques based on the effect promoted by the addition of the surfactants on the fluorescence of the protein tryptophan group. From the dependence of the fluorescence quenching with the protein concentration, the binding isotherms of the surfactants with the protein were determined. The results indicate that CPC is considerably more efficient than DPC in quenching the fluorescence of the tryptophan group. However, even for CPC, eight surfactant molecules must be bound (in average) to each HSA molecule to quench half of the protein intrinsic fluorescence, suggesting an association to zones far away from the locus of the fluorescent moiety. Information regarding the locus of CPC and DPC association to HSA was obtained from their effect on the fluorescence of dansyl derivatives bound to the protein. The results show that: (i) CPC binds to site 2 or near it, quenching the fluorescence of the probe bound to this site and increasing the fluorescence of the probe bound to site 1, and (ii) DPC is less efficient in modifying dansyl derivatives fluorescence, being able to interact weakly with compounds located at both sites 1 and 2.

Selectivity coefficients for the I − /Cl − and I − /Br − ion exchanges at the surfaces of dioctadecyldimethylammonium (large) vesicles were obtained from fluorescence-quenching experiments. The fluorescence of 1-pyrenenonanoic acid incorporated into dioctadecyldimethylammonium chloride and dioctadecyldimethylammonium bromide vesicles is quenched by iodide addition to the intervesicular media

The ion exchange between sodium and a series of alkylammonium cations of the general formula R4−YNHY+ (with R = methyl, ethyl, propyl, butyl; Y = 0 to 4), at the surface of dodecylsulfate micelles, was studied using fluorescence techniques. The selectivity coefficients obtained indicate that all of the organic counterions considered bind more strongly to the micelles than do either sodium or ammonium ions. Replacement of hydrogen by methyl groups (Y = 1 to 3) in the ammonium ion increases the binding strength, the free energy increment for transfer of the counterion from water to the micelle, ΔμO, being −300 ± 30 cal/mole per methyl group. For alkylammonium ions, a ΔμO for transfer of −570 ± 50 cal/mole per methylene group, irrespective of the number of alkyl chains, was found. This indicates that the binding strength of alkylammonium ions is dominated by their hydrophobicity, independent of the spatial distribution of the alkyl groups attached to nitrogen. The possible use of these results to decide between “open” or “structured” micellar models is discussed. Tetraalkyl ions show considerably lower binding affinities than might be expected from the total hydrophobicity of their chains. This result is interpreted in terms of an unfavorable location of the fourth alkyl chain.

The association of Rose Bengal (RB) with bovine serum albumin (BSA) was investigated by absorbance spectroscopy. The binding constant was determined from the effect observed in the absorbance of RB at 548 nm upon addition of the protein according with the Benesi-Hildebrand treatment. Results were obtained in phosphate buffer at pH = 7.0. The effect of the salinity of the buffer and the sensitivity of the binding constant to the presence of urea were also studied. The results obtained allow to conclude that the binding of RB to BSA is dominated by hydrophobic effects

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W=([water]/[AOT])=6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.

A study has been made on the effect of the organic solvent upon the properties of the interface of reverse micelles formed by sodium bis (2-ethylhexyl) sulfosuccinate (AOT) - water. The study has been performed using fluorescence and absorption spectroscopies. The probes considered were 8-anilinonaphtha lene-1-sulfonate (ANS-) and ruthenium tris [(bipyridine)]2+, [Ru (bpy)3]2+, in fluorescence spectroscopy, and E T(30) in absorption spectroscopy. These probes were selected since its spectroscopic behaviour depends on the micro polarity of the medium. Furthermore, they have equal charge (ANS-), opposite charge [Ru(bpy)3]2+ than that of the micelle, or its nature is zwitterionic (E T (30)), which allows for the sensing of the interfacial properties at different depths. The study has been performed at different W = [water]/[AOT] values ranging from ca. 1-2 up to saturation in the external solvent considered. Organic solvents employed were: n-heptane, n-hexane, isooctane, dodecane cyclohexane, benzene, toluene, cumene, chlorobenzene, dichlormethane (DCM), 1,2-dicholoroethane (1,2-DCE) and tetrachloroethylene (TCE), and the results are discussed taking n-heptane as reference solvent. For the three probes considered, n-hexane, isooctane, dodecane and cyclohexane behaves similarly to n-heptane over all the range of W values considered, irrespective of the probe employed. ANS- behaviour in benzene, toluene and TCE is similar to that observed in aliphatic hydrocarbons. Cumene, chlorobenzene, DCM and 1,2 - DCE show a microenvironment more polar than that of aliphatic hydrocarbons. The behaviour of dichloromethane and 1,2 - DCE is similar for the three probes and very different than that observed in aliphatic hydrocarbons. The microenvironment that they offer in front of [Ru(bpy)3]2+ is considerably more polar than that corresponding to n-heptane, while in front of E T(30) is less polar. The results obtained using [Ru(bpy)3]2+ are the more difficult to explain, since displacement towards more polar (in 1,2-DCE and DCM) or less polar (in benzene, cumene, TCE) media were observed

The rate of N-glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by alpha-chymotripsin has been measured in aqueous solutions of cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and dodecyltrimethylammonium bromide at concentrations below and above their critical micellar concentrations (CMC). For the three surfactants considered superactivity was observed, with maximum catalytic efficiencies taking place near the corresponding CMCs. The effect of the surfactants after the CMCs is mostly due to a decreased thermodynamic activity of the substrate due to its incorporation into the micelles. After addition of the surfactants, the Michaelis constant values (corrected to take into account the free substrate concentration) tend to decrease, passing through an ill defined minimum, afterwards reaching a constant value. The catalytic rate constants show the same profiles that the catalytic efficiency, being maxima near the surfactants CMCs. This maximum is more important for the surfactant having the shorter tail. This result is explained by considering that the hydrophobicity of the surfactant influences more the CMC than its association to the enzyme.

The effect of temperature on the oxidation rates of arachidonic acid and linoleic acid incorporated into dipalmitoylphosphatidylcholine liposomes have been investigated. The reaction was started by the photolysis of 2,2'-azobis (2-amidino propane) in order to minimize the dependence of the initiation rate with temperature. Both positive and negative activation energies were obtained for the rate of the oxidation, depending on the amount of oxidable lipid (20% or 50% mol:mol) and the temperature range considered. Changes in the rigidity of the liposomes elicited by temperature were assessed by measuring the generalized polarization of 6-dodecanoyl-2 (dimethylamino naphthalene), Laurdan. The high increase in rate observed at low temperatures employing lipid mixtures that render liposomes in the gel phase is attributed to a reduced rate constant of the termination step of the oxidation chain

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)).

The amount of n-hexanol required to produce a clear microemulsion in a water-in-oil quaternary system comprising a surfactant, 2,2,4-trimethylpentane, water and the alkanol, was evaluated employing cationic, anionic, zwitterionic and neutral surfactants. The critical analytical n-hexanol concentration, the number of n-hexanol molecules at the interface per surfactant (n), and the distribution constant of the alkanol between the organic pseudophase and the interface are determined by the charge of the surfactant head, being almost independent of the alkyl chain length, the counterion and the surfactant head. The values of n are similar for cationic and anionic surfactants. However, due to a less favorable distribution between the organic solvent and the interface, critical n-hexanol concentrations are significantly higher for the anionic surfactants

The effects of addition of alkanols (ethanol, n-hexanol, and 3-ethyl-3-pentanol) on the micropolarity and microviscosity of the head group region in reverse micelles of AOT–heptane–water have been investigated by fluorescence probing methods (ANS fluorescence yield and TMADPH fluorescence anisotropy), complemented by the use of the solvatochromic probe ET(30) in absorption spectroscopy. For all the alkanols considered, ANS fluorescence in AOT reverse micelles (at W=3) is quenched by additive incorporation, being the effect elicited almost independent of the alkanol chain length and topology. As sensed by the ET(30) parameter, the micropolarity of the micelle surface increases, remains unmodified, and decreases upon addition of ethanol, 3-ethyl-3-pentanol, and hexanol, respectively. While ethanol barely modifies the fluorescence anisotropy of TMADPH, 3-ethyl-3-pentanol and n-hexanol addition strongly decrease it. The similarity of the tendencies of ANS data to TMADPH anisotropies and the differences between ANS data and ET(30) values would indicate that, at least for 3-ethyl-3-pentanol and n-hexanol, microviscosity, rather than micropolarity, must be considered to interpret the effect of the alkanols upon the fluorescent behavior of ANS.

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.