INTERACTION OF DIBUCAINEHCl LOCAL ANESTHETICS WITH BACTERIORHODOPSIN IN PURPLE MEMBRANE: A SPECTROSCOPIC STUDY

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Abstract
Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry-DSC) were used to investigate the deprotonation of dibucaine.cntdot.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.cntdot.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (.lambda.max = 568 nm) and deionized (.lambda.max = 608 nm) bR are sensitive to the amount of dibucaine .cntdot. HCl added; (2) the emission spectrum of dibucaine .cntdot. HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.cntdot.HCl in bR was determined as kq = 4.09 .times. 1013 M-1 s-1; (4) the incorporation of dibucaine.cntdot.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 .+-. 1.0.degree. C with 13.6 J/g (3.25 .+-. 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.cntdot.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.