HALOTHANE BINDING TO SOLUBLE-PROTEINS DETERMINED BY PHOTOAFFINITY-LABELING

Background: Recently, halothane and isoflurane have been shown to bind in a saturable manner to serum albumin using NMR and gas chromatography methods. To validate a novel direct photoaffinity labeling method developed in our laboratory, the authors also determined the binding characteristics of halothane to serum albumin, and then extended this approach to other soluble proteins in an initial attempt to understand the interaction of volatile anesthetics and proteins. Methods: Serum albumin (BSA), bacterial luciferase (BL), poly-(L-lysine)(PLL), and poly-(L-glutamate)(PLG) were dissolved in 0.154 M NaCl containing C-14-halothane with or without other volatile anesthetics or ligands, and exposed to 254 nm UV light for 10 s. Covalently bound label was quantitated by scintillation counting after precipitation, filtration, and washing. Binding parameters were calculated by nonlinear least-squares fitting of rectangular hyperbolas or logistic equations. Results: Serum albumin bound halothane in a saturable manner at an apparent K(D) between 0.3 and 0.5 mM. Other volatile anesthetics inhibited binding (K(I), in mM): halothane (0.36), chloroform (1.26), methoxyflurane (2.66), isoflurane (1.47), diethyl ether (45.5), and ethanol (1,040). Oleate and BSA conformational changes (low pH) also inhibited label incorporation. Binding to BL and PLL at pH 7 was nonsaturable and not displaced by unlabeled halothane or the BL substrate decanal. Conversion of PLL to an alpha-helical conformation (pH > 10) increased binding and created a saturable component with an apparent K(D) of 0.55 mM. Alkaline conditions decreased binding to PLG consistent with the loss of alpha-helical domains. Conclusions: Photoaffinity labeling produced results in close agreement with more conventional methods for studying halothane binding, and should be a useful tool for the study of volatile anesthetic binding sites. Halothane binding to soluble proteins depended on their type and conformation, and, in some cases, was saturable within the clinical concentration range, increasing the tenability of discrete proteinaceous sites of action for the inhalational anesthetics.