Fluorescence Anisotropy Measurements under Oxygen Quenching Conditions as a Method to Quantify the Depolarizing Rotations of Fluorophores. Application to Diphenylhexatriene in Isotropic Solvents and in Lipid Bilayers

We have measured the fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) as its fluorescence lifetime is decreased by oxygen quenching. Such studies were done on DPH dissolved in the isotropic solvent mineral oil and for DPH embedded in phospholipid vesicles of either dimyristoyl-l-a-phosphatic'ylcholine (DMPC) or dioleoyl-l-a-phosphatidylcholine (D)PC), each at several temperatures. In order to obtain adequs e quenching increased pressures of oxygen had to be used. Dxygen quenching resulted in significant changes in intens :y and anisotropy, and these effects were reversible. To contr il for possible effects of pressure on the systems under study equivalent experiments were performed with nitrogen, arg n, or helium forming the gas phase. Under these last-mentioi ;d conditions, changes in intensity and anisotropy were insif lificant when compared with those observed with oxygen quenching. The depolarizing rotations of the fluorophore are di scribed by its rotation rate (R) in radians/seconds and its li liting anisotropy at times which are long compared with the f uorescence lifetime, r∞. This latter parameter provides a m asure of the degree to which the fluorophore's environme t hinders its rotational diffusion. Oxygen quenching of flu rescence provides a means to vary the fluorescence lifetiiru simulta: Bous observation of the steady-state fluorescence a lisotropy allows quantitation of both R and For DPH in mineral oil at two different temperatures we found that he values of R obtained from this quenching-anisotropy mea iurement agreed precisely with those obtained from steady-state anisotropy measurements and with the values obtained from differential polarized phase fluorometry (Lakowicz, J. R., et al. (1979) Biochemistry 18 (preceding paper in this issue)). Additionally, r∞ was four to be zero. These results indicate that in mineral oil DH behaves as an ideal unhindered isotropic rotator. In contras DPH embedded in lipid bilayer vesicles of DMPC behaves as an isotropic but highly hindered rotator below the pha; transition temperature, as is indicated by r∞ ≃0.33. Above the phase transition temperature the depolarizing rotations become significantly less hindered, r∞ ≃ 0.03. In DOPC vesicles the depolarizing rotations are unhindered at a temperatures. The temperature profiles of R and r∞ obtaine for DPH in lipid bilayers were in agreement with those ob served using differential polarized phase fluorometr; Quenching-anisotropy measurements of the type we have described provide a powerful method for investigation of time-resolved decays of fluorescence anisotropy without tl direct use of time-resolved methods. The estimation of membrane microviscosity from steady-state anisotrof measurements assumes that the nature of the depolarizin rotations of the fluorophore in the membrane are identical wit those in an isotropic reference solvent. Our results indica' that this assumption is invalid. We estimated the apparei membrane viscosity by three methods: (1) from steady-sta anisotropy measurements; (2) from the rotational rate of DP] within its hindered environment; and (3) from the difftisivii of molecular oxygen. Each method yielded a different value with steady-state polarization giving the highest and oxyge diffusivity the lowest. These results show that any quantitath estimate of microviscosity depends critically upon the mo lecular process used for its estimation.