Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled...

This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of depth in angstroms. Furthermore, the analysis can be extended to quenching by energy-transfer acceptors or brominated probes under appropriate conditions. Application of the method to quenching of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipids by spin-labeled lipids located at three different depths is demonstrated in model membranes. It is shown that the calculated depths of the NBD groups are self-consistent to the extent that they are the same no matter which two spin-labels have been used in a particular experiment. In addition, the calculated depth is independent of spin-label concentration in the membrane within +/- 1 A, ruling out major effects due to spin-label perturbation. The quenching experiments show that the location of the NBD group in head-group-labeled phosphatidylethanolamine is at the polar/hydrocarbon interface and that of an NBD label on the "tail" of cholesterol is deeply buried, as expected. Unexpectedly, NBD labels placed at the end of fatty acyl chains of phosphatidylcholines are also near the polar/hydrocarbon interface. Presumably, the polarity of the NBD group results in "looping" back to the surface of the NBD groups attached to flexible acyl chains.