Diffusion of α-Tocopherol in Membrane Models: Probing the Kinetics of Vitamin E Antioxidant Action by Fluorescence in Real Time

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Abstract
The new fluorescent membrane probe Fluorazophore-L, a lipophilic derivative of the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene, is employed to study the quenching of α-tocopherol (α-Toc) by time-resolved fluorescence in the microheterogeneous environments of Triton XR-100 and SDS micelles, as well as POPC liposomes. Fluorazophore-L has a small nonaromatic fluorescent polar headgroup and an exceedingly long-lived fluorescence (e.g., 140 ns in aerated SDS micelles), which is efficiently quenched by α-Toc (3.9 × 109 M-1 s-1 in benzene). Based on solvatochromic effects and the accessibility by water-soluble quenchers, the reactive headgroup of Fluorazophore-L, along with the chromanol group of α-Toc, resides at the water−lipid interface, which allows for a diffusion-controlled quenching in the lipidic environments. The quenching experiments represent an immobile or stationary case; that is, interparticle probe or quencher exchange during the excited-state lifetime is insignificant. Different quenching models are used to characterize the dynamics and antioxidant action of α-Toc in terms of diffusion coefficients or, where applicable, rate constants. The ideal micellar quenching model is suitable to describe the fluorescence quenching in SDS micelles and affords a pseudo-unimolecular quenching rate constant of 2.4 (± 0.4) × 107 s-1 for a single quencher per micelle along with a mean aggregation number of 63 ± 3. In Triton micelles as well as in unilamellar POPC liposomes, a two-dimensional (lateral) diffusion model is most appropriate. The mutual lateral diffusion coefficient DL for α-Toc and Fluorazophore-L in POPC liposomes is found to be 1.8 (± 0.1) × 10-7 cm2 s-1, about a factor of 2 larger than for mutual diffusion of POPC, but more than 1 order of magnitude lower than a previously reported value. The comparison of the different environments suggests a quenching efficiency in the order benzene ≫ SDS micelles > Triton micelles > POPC liposomes, in line with expectations from microviscosity. The kinetic measurements provide important benchmark values for the modeling of oxidative stress in membranes and other lipidic assemblies. The special case of small lipidic assemblies (SDS micelles), for which the net antioxidant efficacy of α-Toc may be lower than expected on the grounds of its diffusional behavior, is discussed.

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