Cooperative binding of inhaled anesthetics and ATP to firefly luciferase

Firefly luciferase is considered a reasonable model of in vivo anesthetic t argets despite being destabilized by anesthetics, as reflected by different ial scanning calorimetry (DSC). We examined the interaction between two inh aled anesthetics, ATP, luciferase, and temperature, using amide hydrogen ex change, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromo form cause destabilization, as measured by hydrogen exchange, suggesting no nspecific interactions. In the presence of ATP/Mg2+ and at room temperature , the anesthetics produce considerable stabilization with a negative DeltaH , indicating population of a conformer with a specific anesthetic binding s ite. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase exp lain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and an esthetics, suggesting that similar interactions could underlie actions at b iologically relevant targets. (C) 2001 Wiley-Liss, Inc.