Publications (21) View all
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Article: Reply to Comment on "An Interesting Case where Water Behaves as a Unique Solvent. 4-Aminophthalimide Emission Profile to Monitor Aqueous Environment"
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ABSTRACT: Reply to the comments made by Samanta et al, jp-2013-0217j.The Journal of Physical Chemistry B 04/2013; · 3.70 Impact Factor -
Article: An Interesting Case where Water Behaves as a Unique Solvent. 4-Aminophthalimide Emission Profile to Monitor Aqueous Environment.
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ABSTRACT: 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 time-resolved 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 on 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 it was found a time-dependent emission spectrum that shift to the blue with time. Our results show that the solvent-mediated proton transfer process display a fundamental role in the 4-AP emission profile and, for the first time it was proposed a mechanism that fully explain 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 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.The Journal of Physical Chemistry B 01/2013; · 3.70 Impact Factor -
Article: Diffusion of hydrogen peroxide across DPPC large unilamellar liposomes.
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ABSTRACT: The decomposition of hydrogen peroxide catalyzed by catalase entrapped in the pool of dipalmitoylphosphatidyl 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.Chemistry and physics of lipids 07/2012; 165(6):656-61. · 2.15 Impact Factor -
Article: Effect of human serum albumin on the kinetics of 4-methylumbelliferyl-β-D-N-N'-N″ Triacetylchitotrioside hydrolysis catalyzed by hen egg white lysozyme.
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ABSTRACT: 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 Protein Journal 08/2011; 30(6):367-73. · 1.04 Impact Factor -
Article: On the evaluation of the number of binding sites in proteins from steady state fluorescence measurements.
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ABSTRACT: 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.Journal of Fluorescence 04/2011; 21(5):1831-3. · 2.11 Impact Factor
