Publications

Abstract Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/ partition phenomenon was quantified in terms of the partition coefficient (K-p), and/or the equilibrium constant (K-b). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio [ 100: 1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, DeltaH, and indirectly, DeltaG and DeltaS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quanti. cation of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction - possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug.

Abstract 2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper) thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, picosecond time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A ( SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 M potassium mannosylglycerate caused an increase of 7 degreesC in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degreesC and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.

Abstract A novel amperometric glucose sensor was developed based on the facilitated proton transfer across microinterfaces between two immiscible electrolyte solutions. The combination of a 1,3:2,4-dibenzylidene sorbitol/2-nitrophenyl octyl ether gel membrane and 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine as the ionophore allows the transfer of protons from water to the gellified organic phase; the gel membrane is supported on arrays of microholes drilled on a polyester film. The protons are generated as the result of the dissociation of gluconic acid produced during the enzymatic degradation of glucose by glucose oxidase. The characteristics of the glucose sensor were investigated using several experimental conditions, namely, the concentration of ligand and enzyme. The electrochemical response is typical of an enzymatic electrode and displays a linear behavior in the range 0.2-3 mM glucose. The effect of the experimental parameters of the voltammetric technique was also optimized with the aimof improving sensor sensitivity.

Abstract Electrochemical impedance spectroscopy, Fourier transform infrared reflection-absorption spectroscopy, and cyclic voltammetry were employed to characterize polyelectrolyte multilayers (PEMs) fabricated with poly-(styrenesulfonate) as the polyanion and the polypeptides poly-L-histidine, poly-L-lysine, and poly-L-arginine as polycations. The layer-by-layer electrostatic assembly was produced onto alkanethiol-modified gold surfaces. The frequency response reveals that the effect of the number of layers seems to be related to a progressive reduction in the active area of the PEM-modified electrodes. The active area after the deposition of seven layers can be lower than 10% of its original value. The film surface is then inhomogeneous with respect to the transport of the electroactive species and has spots through which transport is quite favored. These structural features of the PEM have been taken into account in the theoretical model of ion transport and very good agreement with the experimental impedance results has been found.

Abstract The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP-HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light-production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low-oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase-catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl-coenzyme A synthetase.

Abstract The standard enthalpy of formation of the 2-amino-3-quinoxalinecarbonitrile-1,4-dioxide compound in the gas-phase was derived from the enthalpies of combustion of the crystalline solid measured by static bomb combustion calorimetry and its enthalpy of sublimation determined by Knudsen mass-loss effusion at T=298.15 K. This value is (383.8+/-5.4) kJ mol(-1) and was subsequently combined with the experimental gas-phase enthalpy of formation of atomic oxygen and with the computed gas-phase enthalpy of formation of 2-amino-3-quinoxalinecarbonitrile, (382.0+/-6.3) kJ mol(-1), in order to estimate the mean (N-O) bond dissociation enthalpy in the gas-phase of 2-amino-3-quinoxalinecarbonitrile-1,4-dioxide. The result obtained is (248.3+/-8.3) kJ mol(-1), which is in excellent agreement with the B3LYP/6-311+G(2d,2p)//B3LYP/631G(d) computed value.

Abstract The standard (pdegrees = 0.1 MPa) molar enthalpy of formation of liquid anthranil was measured at T = 298.15 K by static bomb calorimetry and the standard molar enthalpy of vaporization at T = 298.15 K was obtained using Calvet microcalorimetry. These values were used to derive the standard molar enthalpy of formation of anthranil in the gaseous phase. Thermochemical and quantum chemical comparisons were made to interrelate anthranil and its isomers, 1,2-benzisoxazole, benzoxazole and 2-cyanophenol, and the monocyclic heterocycles, isoxazole and oxazole. Comparisons with benzofurazan and isobenzofuran were also made. Additionally nucleus-independent chemical shifts were used as an aromaticity index. (C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Abstract The standard (p(0) = 0.1 MPa) molar enthalpies of formation, at T= 298.15 K, of crystalline Ni(II), Cu(II) and Zn(II) complexes with N,N'-bis(salicylaldehydo)ethylenediamine (H(2)salen) were determined by solution-reaction calorimetry measurements as, respectively. Delta(f)H(m)(-) {[Ni(salen)], cr}=-(226.1 +/- 3.9)kJmol(-1), DeltafH(m)(o) {[Cu(salen)], cr}=-(139.0 +/- 3.9)kJmol(-1) and Delta(f)H(m)(o) {[Zn(salen)], cr}=-(281.3 +/- 4.6)kJmol(-1). The standard molar enthalpies of sublimation of the same metal complexes, at T=298.15K, were obtained by effusion methods as Delta(cr)(g)H(m)(o), [Ni(salen)]=(163.9+/-3.2)kJmol(-1), Delta(cr)(g)H(m)(o) [Cu(salen)]=(175.3 +/- 2.7)kJmol(-1) and Delta(g)(cr)H(m)(o) [Zn(salen)]=(179.6 +/- 3.7)kJmol(-1). The differences between the mean metal-ligand and hydrogen-ligand bond dissociation enthalpies were derived and discussed in terms of structure, in comparison with identical parameters for complexes of the same metals with other tetradentate Schiff bases involving a N2O2 donor set.

Abstract The standard (pdegrees = 0.1 MPa) molar enthalpies of formation for liquid 2-chloropyrazine and crystalline 2,6-dichloropyrazine, 2,3-dichloroquinoxaline and 2,3,6,7-tetrachloroquinoxatine were derived from the standard molar enthalpies of combustion, in oxygen, to yield CO(2)(g), N(2)(g), and HCl . 600H(2)O(l), at the temperature T = 298.15 K, measured by rotating-bomb combustion calorimetry. The standard molar enthalpies of vaporization or of sublimation, at T = 298.15 K, were measured by Calvet microcalorimetry.

Abstract The mean (N-O) bond dissociation enthalpies were derived for three 2-methyl-3-(R)-quinoxaline 1,4-dioxide (1) derivatives, with R = methyl (1a), ethoxycarbonyl (1b), and benzyl (1c). The standard molar enthalpies of formation in the gaseous state at T = 298.15 K for the three 1 derivatives were determined from the enthalpies of combustion of the crystalline solids and their enthalpies of sublimation. In parallel, accurate density functional theory-based calculations were carried out in order to estimate the gas-phase enthalpies of formation for the corresponding quinoxaline derivatives. Also, theoretical calculations were used to obtain the first and second N-O dissociation enthalpies. These dissociation enthalpies are in excellent agreement with the experimental results herewith reported.