Determination by photoreduction of flip-flop kinetics of spin-labeled stearic acids across phospholipid bilayers.

Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (flip-flop). The flip-flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the flip-flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the flip-flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar flip-flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution kinetics of stearic acid derivatives in biological systems.