Cooperative binding of inhaled anesthetics and ATP to firefly luciferase.
Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific 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 site. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase explain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and anesthetics, suggesting that similar interactions could underlie actions at biologically relevant targets.
Eckenhoff
R G
RG
Department of Anesthesia, University of Pennsylvania Medical Center, Philadelphia 19104-4283, USA. reckenho@mail.med.upenn.edu
Tanner
J W
JW
Liebman
P A
PA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D018685:Anesthetics, Inhalation / Q000378:metabolism*; D005453:Fluorescence; D006221:Halothane / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D019920:Photoaffinity Labels / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D022882:Trihalomethanes / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D018685:Anesthetics, Inhalation; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D022882:Trihalomethanes; D006859:Hydrogen; D014364:Tryptophan; D000255:Adenosine Triphosphate; D008156:Luciferases; C015044:bromoform; D006221:Halothane