Determination of fluid and gel domain sizes in two-component, two-phase lipid bilayers. An electron spin resonance spin label study.

The average sizes of fluid and gel domains in the two-component, two-phase system formed from mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine were determined from an analysis of the electron spin resonance spectral lineshapes of a dimyristoyl phosphatidylcholine-nitroxide spin label as a function of spin label concentration. The ratio, R, of the intensities measured at two magnetic field strengths was found to be diagnostic of a statistical distribution of spin labels in disconnected domains. R is defined as V'/2Vpp, where Vpp is the maximum intensity and V' is the intensity at a position in the wings of a first derivative electron spin resonance line that is a constant multiple of the peak-to-peak linewidth. The intensity ratio for Gaussian or Voigt lineshapes is less than or equal to the value for a Lorentzian lineshape. The intensity ratio was found to be greater than the value for a Lorentzian line when spectra from disconnected domains containing a statistical distribution of spin labels undergoing spin-spin interactions were summed. The intensity ratio, R, calculated by spectral simulations as a function of the average number of labels per domain, N, was found to increase to a maximum with increasing N and then to decrease. The dependence on spin label concentration of the experimentally measured intensity ratios paralleled this predicted behavior. A method is presented to calculate the average number of lipids per fluid or gel domain based on a knowledge of R, and of the distribution of the spin label between the fluid and gel phases determined from the phase diagram. The results demonstrate that the number of lipids per domain increases linearly from a fixed number of nucleation sites, as the fraction of the phase that is disconnected increases. At any given mole fraction of the particular phase, the gel domains are bigger than the fluid domains because they have a lower nucleation density. The results also suggest that the disconnected domains are, in most cases, nonrandomly distributed in the plane of the bilayer.