Vitamin E has been hypothesized to play a role in membrane signalling, organization, and protect the membrane against oxidation. However, these roles are only considered speculation as cellular mechanisms have yet to be identified and very few of Vitamin E’s biophysical properties have been elucidated. Investigating Vitamin E’s partitioning and organization in the bilayer can provide valuable insight on its biological function. Neutron diffraction has demonstrated that alpha-tocopherol (αToc) resides in the cell membrane below the membrane’s lipid-water interface. Further work needs to be done to elucidate its lateral placement in a bilayer. This work aims to investigate αToc’s partitioning preference for a variety of saturated and unsaturated lipid environments. αToc is an innately fluorescent molecule and is the only biologically relevant of the eight vitamers that make up Vitamin E, which is why it is the molecule being used in this study. Utilizing fluorescence lifetimes and fluorescent recovery after quenching we ‘track’ the lipid environment αToc prefers. Fluorescence lifetime measurements are dependent on the environment of the fluorophore. We measure the affinity of αToc for lipids possessing varying degrees of unsaturation relative to a donor 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC) lipid system. Utilizing the auto-fluorescence of αToc, assessment of the quantity of αToc in the DSPC system relative to the lipid of interest (acceptor) is determined by fluorescent life-time measurements. The fluorescent lifetime of αToc differs depending on the lipid environment it resides in. To augment the lifetime measurements, we employ a fluorescence quenching method, where the DSPC donor system will be doped with a 5-doxyl stearic acid quenching agent. Thus, the florescence signal increase as αToc migrates from the donor DSPC system to the acceptor population. This work will identify the interaction of tocopherols, tocotrienols, and their metabolites with different lipid environments, providing a key experimental link between oxidation-sensitive lipids and the affinity of vitamin E for them.

Investigating vitamin E’s lipid partitioning preference by utilizing fluorescent techniques

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