An intramolecular excimer forming probe used to study the interaction of .alpha.-lactalbumin with model membranes

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Abstract
The nonconjugated bichromophoric molecule 1,3-di(1-pyrenyl)propane shows, besides the pyrene monomer fluorescence, a structureless emission due to an intramolecular excited dimer (excimer). In the case of intramolecular excimer forming systems, the ratio of the emission intensities of excimer vs. monomer (IE/IM) is sensitive to changes in membrane structure as will be illustrated here. The present molecule is a useful probe to report on lipid-protein interactions, at least in this model system. It was introduced into the hydrocarbon layer of dimyristoylphosphatidylcholine vesicles in order to study their interaction with .alpha.-lactalbumin (.alpha.-LA) as a function of pH and temperature. Steady-state fluorescence, kinetic, and energy transfer studies, show that, at pH 4, .alpha.-LA strongly interacts with the lipid bilayer. In steady-state fluorescence experiments, changes of the ratio IE/IM and shifts of the transition temperature were observed, reflecting changes of membrane structure caused by interaction with .alpha.-LA. Kinetic studies of the rate of interaction of .alpha.-lactalbumin and measurements of energy transfer from excited tryptophan(s) to the fluorescent probe confirm the steady-state experiments. These results agree with previously reported microcalorimetric, gel chromatogrpahic, and fluorescence polarization studies.