Binding of the Volatile Anesthetic Chloroform to Albumin Demonstrated Using Tryptophan Fluorescence Quenching

Abstract

The site(s) of action of the volatile general anesthetics remain(s) controversial, but evidence in favor of specific protein targets is accumulating. The techniques to measure directly volatile anesthetic binding to proteins are still under development. Further experience with the intrinsic protein fluorescence quenching approach to monitor anesthetic-protein complexation is reported using chloroform. Chloroform quenches the steady-state tryptophan fluorescence of bovine serum albumin (BSA) in a concentration-dependent, saturable manner with a K d ‫؍‬ 2.7 ؎ 0.2 mM. Tryptophan fluorescence lifetime analysis reveals that the majority of the quenching is due to a static mechanism, indicative of anesthetic binding. The ability of chloroform to quench BSA tryptophan fluorescence was decreased markedly in the presence of 50% 2,2,2-trifluoroethanol, which causes loss of tertiary structural contacts in BSA, indicating that protein conformation is crucial for anesthetic binding. Circular dichroism spectroscopy revealed no measurable effect of chloroform on the secondary structure of BSA. The results suggest that chloroform binds to subdomains IB and IIA in BSA, each of which contains a single tryptophan. Earlier work has shown that these sites are also occupied by halothane. The present study therefore provides experimental support for the theory that structurally distinct general anesthetics may occupy the same domains on protein targets.

Anesthetic-like Interactions of Nitric Oxide with Albumin and Hemeproteins

“…Recent studies provide support for interactions of both halothane and chloroform with tryptophans of HSA and bovine serum albumin (13,14). Considerations of the bonding interactions involved and the frequencies of NNO-IR bands make tryptophan an attractive binding site for both NNO and NO.…”

“…Specific, saturable binding of halothane and other anesthetics to bovine serum albumin and anesthetic-induced alterations in the structure or carrier function of this protein have been shown (11)(12)(13)(14)(15)(16). The anesthetic nitrous oxide (NNO) was detected at sites within human serum albumin (HSA) (5).…”

Spectroscopic Studies of Interaction of Protein with Cerium Oxide Nanoparticles

“…The fluorescence decays of BSA were fitted with two exponentials, T1 = 6.50 ns and T2 = 2.46 ns and is consistent with the studies that lifetimes of tryptophan fluorescence are often multi exponential [28]. The longer and shorter lifetimes indicated that BSA contained two tryptophan residues that fluoresced in two different environments [29] and one of the tryptophan residues in the protein may be buried inside the hydrophobic interior of protein whereas the other tryptophan residue may be close to quencher [30]. This is in good agreement with the reports that BSA has two tryptophan residues, Trp-134 in the first domain located on the surface of molecule and Trp-212 in the second domain located within a hydrophobic binding pocket [14].…”

Studies on the Interaction of Cefepime Hydrochloride with Bovine Serum Albumin by Fluorescence, Synchronous Fluorescence, Three-Dimensional Fluorescence and Circular Dichroism

“…Fluorescence and synchronous fluorescence spectroscopic studies of bovine serum albumin: In addition to the proximity of bound cefepime hydrochloride to Trp residue, fluorescence quenching might result from structural change of BSA upon cefepime hydrochloride binding [42]. As is well known, the maximum fluorescence emission wavelength (λ em ) of Trp residues is closely related to the polarity of the microenvironment around Trp residues.…”