Amino Acid Resolution of Halothane Binding Sites in Serum Albumin

Abstract

Saturable binding of various inhaled anesthetics to serum albumin has been shown with a variety of approaches. In order to determine the location of halothane binding sites in serum albumin, both human and bovine serum albumins (HSA and BSA) were photolabeled with [ 14 C]halothane, and subjected to proteolysis and microsequencing. BSA was found to have a higher affinity for halothane than HSA, and it contained two specifically labeled sites. One site was characterized by diffuse labeling from Trp 212 -Leu 217, and the other by a more discrete and higher affinity labeling at Trp 134 -Gly 135. HSA contained only a single labeled site, and although lower affinity, was determined to be analogous to BSA Trp 212 . The position 130 -140 region of HSA, having a leucine instead of tryptophan at position 134, was not labeled. These results demonstrate specific and discrete binding of an inhaled anesthetic to a mammaliansoluble protein, and further suggest the importance of aromatic residues as one feature of inhaled anesthetic binding sites.Volatile inhalational anesthetics can bind to and alter the function of a variety of proteins, both soluble and membranebound (1). Determination of the characteristics of these binding sites in proteins should allow prediction of the interactive forces producing binding (immobilization) and also hint at the structural or dynamical consequences to the target. Furthermore, the demonstration of a binding motif common to many protein targets will simplify the search for anesthetic binding domains in other systems. Unfortunately, the character of anesthetic binding sites in protein has been difficult to determine directly, and few examples are available.While consensus places important actions of anesthetics at membrane protein, the presence of lipid and multiple apparently specific binding sites (2) renders these complex models presently unsuitable for a focused search of binding site features. Certain soluble proteins, on the other hand, appear to have a limited number of discrete binding sites for inhalational anesthetics, making them reasonable initial candidates for characterizing anesthetic binding domains. Accordingly, several approaches have identified specific, saturable binding of halothane in bovine serum albumin (3-6) and have also demonstrated anesthetic-induced alterations in the structure or carrier function (7)(8)(9)(10)(11)(12) C]2-bromo-2-chloro-1,1,1-trifluoroethane; 6.6 mCi/mmol) was purchased from DuPont NEN as the neat compound, which was dissolved in buffer as a stock 6 mM solution and stored at Ϫ80°C. Electrophoresis reagents and supplies were purchased from Bio-Rad.Photoaffinity Labeling-Albumin solutions (generally 15 M) in deoxygenated (argon-bubbled) 150 mM NaCl, containing various concentrations (0.05-0.4 mM) of [ 14 C]halothane was exposed to 254 nm light (Oriel Hg pencil calibration lamp at 7 mm) for 60 -100 s in 5-mm path-length quartz cuvettes (2 ml) at 20 -22°C. The final addition completely filled the cuvette, which was then sealed with a Teflon stopper, ...

Multiple Specific Binding Targets for Inhaled Anesthetics in the Mammalian Brain

“…Finally, photolabeling is a nonequilibrium binding method, and we have used it here to estimate some equilibrium binding parameters for the different protein bands. Again, the estimates of affinity derived from these studies agree with those obtained by equilibrium methods in purified protein models like serum albumin (Johansson et al, 1995;Eckenhoff, 1996a), sometimes being lower by a factor of 2 to 3. Thus, our photolabeling methodology should be reliably reporting both stoichiometry and affinity values for these samples of brain protein.…”

“…We have previously shown that halothane, a clinical inhaled anesthetic, is a reasonable photoaffinity probe capable of providing residue-level resolution of halothane binding location in a protein matrix (Eckenhoff and Shuman, 1993;Eckenhoff, 1996a). Using this approach in rat brain slices, we have also found widespread incorporation of label .…”

Fluorescence study of Bovine Serum Albumin and Ti and Sn Oxide Nanoparticles Interactions.

“…It has been reported that trifluoroacetic acid binds strongly to BSA 13 . Similar compounds such as halothane (a trifluorocarbon compound) have also high affinity in two binding sites where the two tryptophan aminoacid residues are located in BSA structure 14 . TFAc is also described as a strong hydrogen bond donor 15 , and does not affect tryptophan or tryptophan derivatives fluorescence 16 .…”

Inhaled Anesthetic Binding Sites in Human Serum Albumin

“…We have previously reported that bovine serum albumin binds halothane with dissociation constants of about 1 mM and that human serum albumin binds with about 3-5-fold lower affinity (4,5). Photolabeling and fluorescence quenching allowed localization of halothane binding to the immediate vicinity of the two tryptophan residues of bovine serum albumin (Trp-214 and Trp-135).…”