New method for determining tie-lines in coexisting membrane phases using spin-label ESR.
A full description of coexisting phases includes their respective compositions, which are provided by the thermodynamic tie-lines. Fluorescence microscopy enables visualization of coexisting lipid phases in giant unilamellar phases, but the composition information is missing. For cholesterol-containing lipid mixtures, knowledge of the compositions of the coexisting phases is important for understanding the nature of "membrane rafts". We propose and demonstrate a new method, based on ESR spectroscopy, for determining tie-lines in regions of two-phase coexistence in a ternary lipid mixture. Over 100 different lipid compositions containing the spin-labeled phospholipid 16-PC in or near the two-phase coexistence region of the liquid-disordered and the gel phases of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) were studied to determine five tie-lines, spread over virtually the full range of this coexistence region. The method is based on the facts that (1) along a tie-line the ESR spectrum must be a superposition of the two ESR spectra from the respective single phases at the phase boundaries (connected by the tie-line) in a ratio given by the lever rule; (2) along a tie-line the partition coefficient, K(p), for the spin-label, which is also determined in our method, must be constant. We do find that K(p) for 16-PC is close to unity, but its value depends on the particular tie-line. The coexisting phases in equilibrium are characterized by the K(p) of the spin-label and its respective dynamic parameters obtained from fitting the ESR spectra to dynamical models.
Chiang
Yun-Wei
YW
Baker Laboratory of Chemistry and Chemical Biology, and National Biomedical ACERT Center for Advanced ESR Technology, Cornell University, Ithaca, New York 14853-1301, USA.
Zhao
Jiang
J
Wu
Jing
J
Shimoyama
Yuhei
Y
Freed
Jack H
JH
Feigenson
Gerald W
GW
D002784:Cholesterol / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008055:Lipids / Q000737:chemistry*; D008567:Membranes, Artificial; D044367:Phase Transition
D008055:Lipids; D008567:Membranes, Artificial; D002784:Cholesterol