RESUMO
The solubilization of biological membranes by detergents has been used as a major method for the isolation and purification of membrane proteins and other constituents. Considerable interest in this field has resulted from the finding that different components can be solubilized selectively. Certain membrane constituents are incorporated into small micelles, whereas others remain in the so-called detergent-resistant membrane domains that are large enough to be separated by centrifugation. The detergent-resistant fractions contain an elevated percentage of cholesterol, and thus its interaction with specific lipids and proteins may be key for membrane organization and regulation of cellular signaling events. This report focuses on the solubilization process induced by the sucrose monoester of myristic acid, beta-D-fructofuranosyl-6-O-myristyl-alpha-D-glucopyranoside (MMS), a nonionic detergent. We studied the effect of the head group and the cholesterol content on the process. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and dioctadecyl-dimethyl-ammonium chloride (DODAC) vesicles were used, and the solubilization process was followed using Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) generalized polarization (GP) measurements, carried out in the cuvette and in the 2-photon microscope. Our results indicate that: (i) localization of the MMS moieties in the lipid bilayer depends on the characteristics of the lipid polar head group and influences the solubilization process. (ii) Insertion of cholesterol molecules into the lipid bilayer protects it from solubilizaton and (iii) the microscopic mechanism of solubilization by MMS implies the decrease in size of the individual liposomes.
Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Ácido Mirístico/química , Fosfolipídeos/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Detergentes/química , Cinética , Lauratos/química , Lipossomos/síntese química , Lipossomos/química , Fosfatidilcolinas/química , Compostos de Amônio Quaternário/química , Solubilidade , Espectrometria de FluorescênciaRESUMO
Detection of O(2)(1Delta(g)) phosphorescence emission, lambda(max)=1270 nm, following laser excitation and steady state methods were employed to determine the total rate constant, k(T), for the reaction between the non-steroidal anti-inflammatory drug piroxicam (PRX) and singlet oxygen in several solvents. Values of k(T) ranged from 0.048+/-0.003 x 10(6) M(-1) s(-1) in chloroform to 71.2+/-2.2 x 10(6) M(-1) s(-1) in N,N-dimethylformamide. The chemical reaction rate constant, k(R), was determined by using thermal decomposition of 1,4-dimethylnaphthalene endoperoxide as the singlet oxygen source. In acetonitrile, the k(R) value is equal to 5.0+/-0.4 x 10(6) M(-1) s(-1), very close to the k(T) value. This result indicates that, in this solvent, the chemical reaction corresponds to the main reaction path. Dependence of total rate constant on the solvent parameters pi* and beta can be explained in terms of a reaction mechanism that involves the formation of a perepoxide intermediate. Rearrangement of the perepoxide to dioxetane followed by ring cleavage and transacylation accounts for the formation of N-methylsaccharine and N-(2-pyridyl)oxamic acid, the main reaction products. Data obtained in dioxane-water (pH 4) mixtures with neutral enolic and zwitterionic tautomers of piroxicam in equilibrium show that the zwitterionic tautomer reacts with singlet oxygen faster than the enolic tautomer.
Assuntos
Anti-Inflamatórios não Esteroides/química , Dimetilformamida/química , Piroxicam/química , Oxigênio Singlete/química , Estrutura Molecular , SolventesRESUMO
Proteins bearing colored prosthetic groups, such as the heme group in hemoglobin or the bilin group in c-phycocyanin, quench singlet oxygen by interactions at the apoprotein and the prosthetic group levels. In both proteins, chemical modification of the chromophore constitutes only a minor reaction pathway. While total deactivation of singlet oxygen takes place with rate constants of 4.0 x 10(9) and 4.2 x 10(8) M-1 s-1 for hemoglobin and phycocyanin, respectively, the bleaching of the chromophore takes place with rate constants of 3.2 x 10(6) and approximately 1 x 10(7) M-1 s-1. Irradiation of phycocyanin with red light bleaches the chromophore with low yields (approximately 0.8 x 10(-4)). Part of this bleaching is mediated by singlet oxygen produced by the irradiation of the bilin group. The low relevance of the singlet oxygen pathway is compatible with a low quantum yield (approximately 10(-3)) of free singlet oxygen production after irradiation of the protein.