Background
Although it has been suggested that anesthetics alter protein conformational states by binding to nonpolar sites within the interior regions of proteins, the rate and extent to which anesthetics penetrate membrane proteins has not been characterized. The authors report the use of steady-state and stopped-flow spectroscopy to characterize the interactions of halothane with receptor membranes.
Methods
Steady-state and stopped-flow fluorescence spectroscopy was used to characterize halothane quenching of nicotinic acetylcholine receptor (nAcChoR)-rich membrane intrinsic fluorescence and the rate of isoflurane-induced nAcChoR desensitization.
Results
At equilibrium, halothane quenched only 54 +/- 1.4% of all tryptophan fluorescence. Diethyl ether failed to reduce fluorescence quenching by halothane, suggesting that it does not bind to the same protein sites as halothane. Stopped-flow fluorescence traces defined two kinetic components of quenching: a fast component that occurred in less than 1 ms followed by a slower biphasic fluorescence decay. Protein unfolding with sodium dodecyl sulfate reduced halothane's Stern-Volmer quenching constant, eliminated the biphasic decay, and rendered fluorescence accessible to quenching by halothane within 1 ms. Functional studies indicate that anesthetic-induced desensitization of nAcChoR occurs in less than 2 ms.
Conclusions
Unquenchable fluorescence arises from tryptophan residues that are buried within the protein and protected from halothane. Sodium dodecyl sulfate unfolds membrane proteins and allows previously buried fluorescence protein residues to be rapidly and homogeneously quenched by halothane. Halothane quenches protein components of nAcChoR membranes over the same concentration range and time scale that it exerts its functional effects, a finding that is generally consistent with a protein site of action.
The solubility of chlorophenols as affected by surfactant was investigated. Three kinds of surfactant, sodium dodecyl sulfate, Triton X-100, and Brij 35, were utilized. The solubilization of chlorophenols by surfactant follows the order of 2,4,6-trichlorophenol > 2,4-dichlorophenol > 2,6-dichlorophenol > 2-chlorophenol; and the critical micelle concentration is an important index. The adsorption reactions of 2,4-dichlorophenol and 2,4,6- trichlorophenol onto hydrous montmorillonite in the presence of surfactant were examined. The presence of surfactant decreased the adsorption of chlorophenols significantly. The roles of hydrophobicity of chlorophenols in solubilization and adsorption behaviors are discussed.
Different submicellar solubilization mechanisms of two systems, Triton X-100/tetradecane and sodium dodecyl sulfate (SDS)/butyl methacrylate, are revealed on the molecular scale by 1H NMR spectroscopy and 2D diffusion ordered spectroscopy (DOSY).
Interaction of sodium dodecyl sulfate with multi-subunit proteins. A case study with carmin