19F-NMR spin-spin relaxation (T2) method for characterizing volatile anesthetic binding to proteins. Analysis of isoflurane binding to serum albumin.
This paper characterizes the low-affinity ligand binding interactions of a fluorinated volatile anesthetic, isoflurane (CHF2OCHClCF3), with bovine serum albumin (BSA) using 19F-NMR transverse relaxation (T2). 19F-NMR spectra of isoflurane in aqueous BSA reveal a single isoflurane trifluoromethyl resonance, indicative of rapid exchange of isoflurane between protein-bound and aqueous (free) environments. The exchange is slow enough, however, that the chemical shift difference between bound and free isoflurane (delta omega = 0.545 ppm) contributes to the observed isoflurane T2. The contribution of delta omega to T2 can be minimized by shortening the interval between 180 degrees refocusing pulses in the Carr-Purcell-Meiboom-Gill pulse sequence used to monitor T2. Analysis of the dependence of T2 on interpulse interval additionally allows determination of the T2 (6.2 ms) and the average lifetime (tau b = 187 microseconds) of bound isoflurane molecules. By use of a short interpulse interval (less than 100 microseconds), T2 measurements can readily be used to analyze equilibrium binding of isoflurane to BSA. This analysis revealed a discrete saturable binding component with a KD = 1.4 mM that was eliminated either by coincubation with oleic acid (6 mol/mol of BSA) or by conversion of BSA to its "expanded" form by titration to pH 2.5. The binding was independently characterized using a gas chromatographic partition analysis (KD = 1.4 mM, Bmax = 3-4 sites). In summary, this paper describes a method whereby T2 measurements can be used to characterize equilibrium binding of low-affinity ligands to proteins without the confounding contributions of chemical shift.(ABSTRACT TRUNCATED AT 250 WORDS)
Dubois
B W
BW
Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Evers
A S
AS
D000777:Anesthetics / Q000378:metabolism*; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002417:Cattle; D002849:Chromatography, Gas; D005461:Fluorine; D007530:Isoflurane / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism*
D000777:Anesthetics; D001798:Blood Proteins; D012710:Serum Albumin, Bovine; D005461:Fluorine; D007530:Isoflurane