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.
A Stopped-Flow Kinetic Study of the Solubilization of Acridine Orange and Its 10-Alkyl Derivatives into the Micelle of Sodium Dodecyl Sulfate
