Bound volatile general anesthetics alter both local protein dynamics and global protein stability

Background: Recent studies have demonstrated that volatile general anesthet ic agents such as halothane and isoflurane may bind to discrete sites on pr otein targets, In the case of bovine serum albumin, the sites of halothane and chloroform binding have been identified as being located in the IB and ITA subdomains. This structural Information provides a foundation for more detailed studies into the potential mechanisms of anesthetic action. Methods: The effect of halothane and isoflurane and the nonimmobilizer 1,2- dichlorohexafluorocyclobutane on the mobility of the indole ring in the try ptophan residues of albumin was investigated using measurements of fluoresc ence anisotropy, Myoglobin served as a negative control. In addition, the e ffect of bound anesthetic agents on global protein stability was determined by thermal denaturation experiments using near-ultraviolet circular dichro ism spectroscopy. Results: The fluorescence anisotropy measurements showed that halothane and isoflurane decreased the mobility of the indole rings In a concentration-d ependent manner. The calculated dissociation constants were 1.6 +/- 0.4 and 1.3 +/- 0.3 mM for isoflurane and halothane, respectively. In contrast, bo th agents failed to increase the fluorescence anisotropy of the tryptophan residues in myoglobin, compatible with lack of binding. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane caused no change in the fluorescence anis otropy of albumin. Binding of the anesthetic agents stabilized the native f olded form of albumin to thermal denaturation, Analysis of the thermal dena turation data yielded dissociation constant values of 0.98 +/- 0.10 mM for isoflurane and 1.0 +/- 0.1 mM for halothane. Conclusions: Attenuation of local side-chain dynamics and stabilization of folded protein conformations may represent fundamental modes of action of v olatile general anesthetic agents. Because protein activity is crucially de pendent on inherent flexibility, anesthetic-induced stabilization of certai n protein conformations may explain how these important clinical agents cha nge protein function.