Th. Huang et al., A C-13 AND H-2 NUCLEAR-MAGNETIC-RESONANCE STUDY OF PHOSPHATIDYLCHOLINE CHOLESTEROL INTERACTIONS - CHARACTERIZATION OF LIQUID-GEL PHASES, Biochemistry, 32(48), 1993, pp. 13277-13287
A detailed study on the structure, dynamics, and thermodynamic behavio
r of phosphatidylcholine/cholesterol (PC/CHOL) mixtures was undertaken
using differential scanning calorimetry (DSC) and solid-state nuclear
magnetic resonance (NMR) spectroscopy. DSC thermograms of mixtures of
cholesterol (CHOL) with 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC)
, 1,2-distearoyl-sn-phosphatidylcholine (DSPC), and 1,2-diarachidoyl-s
n-phosphatidylcholine (DAPC) showed a broadening of the first-order ge
l --> liquid crystalline transition and a decrease in the transition e
nthalpy, indicating a gradual loss of cooperativity for high CHOL conc
entrations. DPPC and DSPC were labeled with C-13 at the carbonyl group
of the sn-2 chain and H-2 was introduced into the middle of the sn-2
chain at the 6- and 12-position for DPPC and DSPC, respectively. The C
-13 and H-2 NMR spectra of each labeled lipid were studied as a functi
on of temperature and CHOL concentration. The residual quadrupole spli
tting in the H-2 NMR spectra, DELTAnu(Q perpendicular-to), was analyze
d as a function of temperature and composition. For CHOL concentration
s less than 30 mol %, a precipitous change in DELTAnu(Q perpendicular-
to) occurs near the chain melting temperature of the phospholipid. Fur
ther increases in CHOL concentration broaden the transition and shift
the midpoint to higher temperature, indicating the presence of a new p
hase at higher CHOL contents. Moreover, at a given temperature, DELTAn
u(Q perpendicular-to) increases with increasing cholesterol content, w
hich indicates a more ordered structure. The C-13 NMR spectra in the g
el state consisted of a superposition of two components which can be a
ttributed to both gel-like and fluid phospholipid domains in the bilay
er. This two-component spectrum can be simulated quantitatively with a
two-parameter chemical exchange model, which permits the fraction of
each form and the exchange rate to be determined as a function of temp
erature and composition. At high CHOL contents the line width of the f
luid component broadens, suggesting an increase in the exchange rate b
etween the domains. These results were interpreted in terms of a tempe
rature composition diagram with one region L(beta'), two regions LG(I)
and LG(II), and one liquid crystalline region L(alpha), with LG denot
ing ''liquid-gel'' type phases. Liquid-gel phases correspond to phases
with increased order in the hydrocarbon chains (in comparison to that
of the pure PC bilayer in the L(alpha) phase) combined with fast limi
t axial diffusion that averages the C-13 NMR spectrum to a ''fluidlike
'' line. These phases are similar to those found in phosphatidylethano
lamine/cholesterol (PE/CHOL) mixtures [Blume, A., & Griffin, R. G. (19
81) Biochemistry 24, 6230] and are in agreement with the results of Vi
st and Davis [(1990) Biochemistry 29, 451].