The parallax method is a method by which the depth of fluorescent molecules within a membrane is calculated from the ratio of quenching induced by two spin-labeled phospholipids at different depths. In this report, the method is extended to measurements of depth in the polar headgroup region of the membrane through use of a lipid with a spin-label attached to the polar choline moiety. Quenching data indicate that the choline-attached nitroxide is close to 19.5 angstrom from the bilayer center, in good agreement with the choline location previously determined by diffraction measurements. By using quenching results obtained with this polar headgroup-labeled phospholipid, depths more accurate than those measured previously can be obtained for fluorophores in the polar region of the membrane. It appears that the most reliable results are obtained when depth is calculated from the quenching of the two spin-labels that quench a specific fluorophore most strongly. Applying this approach to a series of anthroyloxy-labeled fatty acids indicates that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group. The fatty acid carboxyl group itself is close to 18.6 angstrom from the bilayer center in the ionized form and 16 angstrom from bilayer center in the protonated form. This is close to the depth of the carboxyl groups on phospholipid fatty acyl chains. More accurate depths have also been obtained for 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled phospholipids using the quenching of the choline-attached spin-label. The NBD group appears to locate about 19-20 angstrom from the bilayer center, in agreement with energy-transfer results (Wolf et al. (1992) Biochemistry 31, 2865-2873). Spin-label quenching has also been used to show that the anthroyloxy groups move toward the surface of the bilayer while in the excited state. The depth of anthroyloxy groups immediately after excitation appears to be given by the depth measured at the shortest emission wavelengths.
