CALIBRATION OF THE PARALLAX FLUORESCENCE QUENCHING METHOD FOR DETERMINATION OF MEMBRANE PENETRATION DEPTH - REFINEMENT AND COMPARISON OF QUENCHING BY SPIN-LABELED AND BROMINATED LIPIDS

We previously introduced the "parallax" method, which uses fluorescence quenching by spin-labeled lipids in order to measure the depth of molecules within a membrane [Chattopadhyay, A., & London, E. (1987) Biochemistry 26, 39-45]. In this report the accuracy of this method is established by comparison of spin-label quenching to that obtained using brominated lipids. To accomplish this, the fluorescent molecules used were a fatty acid labeled with a carbazole buried deeply within the acyl chain region of the membrane, an acyl-Trp with the Trp residue residing near the polar membrane region, and cytochrome b5, which has Trp residues in its membrane-inserted region. The depths calculated from the amount of bromine quenching agreed with those determined using parallax analysis. This indicates that the depth reported by parallax analysis is accurate and that the spin labels reside very close to their predicted locations in the membrane. Furthermore, there was good agreement when parallax analysis was applied both to quenching by brominated and spin-labeled molecules, suggesting that the analysis is valid in both cases. The effect that different distributions and motions of fluorophores and quenchers would have on parallax analysis was also examined. For uniform distributions of quenchers or fluorophores over a range of depths, it was found that the analysis reports the average fluorophore depth. In addition, experimental data suggest that motional effects do not significantly alter the measured depths. This is consistent with the motions during the short excited state lifetime of the fluorophores being relatively small and/or relatively isotropic.