Binding of the active metabolite of chloral hydrate, 2,2,2-trichloroethanol, to serum albumin demonstrated using tryptophan fluorescence quenching.

Chloral hydrate, a sedative/hypnotic agent widely used in the pediatric population, is converted to the active metabolite 2,2,2-trichloroethanol (TCE) in the liver. Tryptophan fluorescence quenching has been used previously to show that halothane and chloroform bind saturably to serum albumin, and a similar approach is used here to demonstrate that TCE also binds to albumin. TCE quenches the steady-state tryptophan fluorescence of bovine serum albumin (BSA) in a concentration-dependent, saturable manner with a K(D) = 3.3 +/- 0.3 mmol/l. Unlike halothane and chloroform, however, TCE also elicits a concentration-dependent blue-shift in the fluorescence emission spectrum of BSA and human serum albumin. This indicates that TCE induces a conformational change in the protein, causing the tryptophan to experience a change in its chemical environment, thus shifting the peak of the emission spectrum. Circular dichroism spectroscopy revealed a decrease in the alpha-helical content of BSA from 65.8 +/- 0.4 to 62.9 +/- 0.6% when TCE was present at a concentration of 30 mmol/l, providing further evidence for a conformational change. There is evidence that TCE potentiates the action of ligand-gated ion channels such as the GABA(A) and 5-HT(3) receptors, and the present results suggest that anesthetic alcohols may act by binding to these proteins and inducing structural changes that may in turn alter protein function.