Determining ethanol distribution in phospholipid multilayers with MAS-NOESY spectra.

The location of an ethanol molecule within a membrane, an issue of considerable controversy, was investigated directly by NMR with two-dimensional NOESY. Lipid and ethanol 1H NMR resonances of multilamellar liposomes were resolved by magic-angle spinning (MAS). We observed strong proton lipid-ethanol crosspeaks in dispersions of saturated dimyristoylphosphatidylcholine and monounsaturated stearoyloleoylphosphatidylcholine and in polyunsaturated stearoyldocosahexaenoylphosphatidylcholine. Crosspeak intensity has been interpreted in terms of an ethanol distribution function over the lipid bilayer. Ethanol resides with the highest probability at the lipid water interface near the lipid glycerol backbone and upper methylene segments of lipid hydrocarbon chains. Chain unsaturation has only a minor influence on the ethanol distribution function. In all cases, the ethanol concentration in the bilayer core is significantly lower. At ambient temperature all lipid-ethanol crosspeaks are positive. Crosspeak intensity decreases with increasing water content and increasing temperature most likely because of shorter correlation times of lipid and ethanol reorientation. This suggests a lifetime for specific lipid-ethanol contacts of about 1 ns. Lipid-ethanol and lipid-lipid crosspeaks reflect the high degree of motional disorder of lipids and incorporated ethanol in membranes and the rather arbitrary nature of the location of the lipid-water interface.

Nuclear magnetic resonance investigation of hydrocarbon chain packing in bilayers of polyunsaturated phospholipids.

2H nuclear magnetic resonance (NMR) on chain-deuterated phospholipids has been used to study the influence of the degree of unsaturation on lipid chain packing and on area per molecule at the lipid water interface. Order and motions of deuterated stearic acid in position sn-1 of phosphatidylcholines (PC) containing 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3, or 22:6n-3 in position sn-2 were investigated in pure PC and in mixtures of PC in a phosphatidylethanolamine (PE) matrix. Results reveal that lipid packing in bilayers is mainly controlled by packing requirements at the lipid water interface. Increasing degrees of unsaturation lower chain order and increase area per PC molecule, whereas inclusion of PE in model membranes has the opposite effect. Chain order and motions in highly unsaturated lipid membranes are less sensitive to changes in temperature. Temperature sensitivity decreases further upon incorporation of PC into a PE matrix. Unsaturation induces chain disordering, which may be interpreted as an increase in area per molecule of lipids toward the center of the bilayer. This may result in a lower packing density of unsaturated lipids at the lipid water interface. We hypothesize that these differences in lipid packing and dynamics may influence activity of membrane proteins.

2H nuclear magnetic resonance order parameter profiles suggest a change of molecular shape for phosphatidylcholines containing a polyunsaturated acyl chain.

Solid-state 2H nuclear magnetic resonance spectroscopy was used to determine the orientational order parameter profiles for a series of phosphatidylcholines with perdeuterated stearic acid, 18:0d35, in position sn-1 and 18:1 omega 9, 18:2 omega 6, 18:3 omega 3, 20:4 omega 6, 20:5 omega 3, or 22:6 omega 3 in position sn-2. The main phase transition temperatures were derived from a first moment analysis, and order parameter profiles of sn-1 chains were calculated from dePaked nuclear magnetic resonance powder patterns. Comparison of the profiles at 37 degrees C showed that unsaturation causes an inhomogenous disordering along the sn-1 chain. Increasing sn-2 chain unsaturation from one to six double bonds resulted in a 1.6-kHz decrease in quadrupolar splittings of the sn-1 chain in the upper half of the chain (or plateau region) and maximum splitting difference of 4.4 kHz at methylene carbon 14. The change in chain order corresponds to a decrease in the 18:0 chain length of 0.4 +/- 0.2 A with 18:2 omega 6 versus 18:1 omega 9 in position sn-2. Fatty acids containing three or more double bonds in sn-2 showed a decrease in sn-1 chain length of 0.7 +/- 0.2 A compared with 18:1 omega 9. The chain length of all lipids decreased with increasing temperature. Highly unsaturated phosphatidylcholines (three or more double bonds in sn-2) had shorter sn-1 chains, but the chain length was somewhat less sensitive to temperature. The profiles reveal that the sn-1 chain exhibits a selective increase in motional freedom in a region located toward the bottom half of the chain as sn-2 unsaturation is increased. This corresponds to an area increase around carbon atom number 14 that is three to four times greater than the increase for the top part of the chain. A similar asymmetric decrease in order, largest toward the methyl end of the chain, was observed when 1 -palmitoyl-2-oleoylphosphatidylethanolamine goes from a lamellar to an inverse hexagonal (H,,) phase. This is consistent with a change to a more wedge-shaped space available for the acyl chain.

Role of interactions at the lipid-water interface for domain formation.

The lipid-water interface is critical for the packing of lipid molecules in membranes. We have demonstrated that lateral phase separation in membranes can be driven by electrostatic interactions such as those involving charged lipid species and oppositely charged peptides, in addition to hydration effects at the lipid-water interface. By using nuclear magnetic resonance (NMR), circular dichroism and fluorescence spectroscopy we have shown that binding of a 21-amino acid peptide containing six positively charged arginine residues to mixed phosphatidylcholine (PC)/phosphatidylglycerol (PG) membranes results in a conformational change in the peptide from a random coil to a helical structure and causes the formation of domains of negatively charged PG. Binding of the peptide to PG membranes disorders the lipid hydrocarbon chains. The strength of lipid-peptide binding at the interface, the conformational change in the peptide, and domain formation with the negatively charged lipid are coupled energetically. The lipid-peptide association constant is lower for membranes containing 20 mol% PG in PC/PG mixtures than for 100% PG membranes. We suggest that one of the factors that lower the association constant in PC/PG membranes is entropic energy of formation of PG domains. Besides electrostatic interactions, hydration of lipids is important for domain formation. We have shown that dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine separate under conditions of decreased water activity. Furthermore, water activity controls lipid packing stress in the hydrocarbon core and the headgroups of membranes as demonstrated by induction of an inverse-hexagonal-to-lamellar phase transition in dioleoylphosphatidylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparative high-performance liquid chromatography purification of polyunsaturated phospholipids and characterization using ultraviolet derivative spectroscopy.

A preparative reversed-phase HPLC system utilizing an isocratic mobile phase to purify up to 10-mg quantities of phospholipids is described. The method was developed to separate oxidation products of polyunsaturated phospholipids from intact, parent lipids. The method is useful for phosphatidylcholine and phosphatidylethanolamine on a preparative scale and for phosphatidylserine and phosphatidic acid on an analytical scale. Both intact phospholipids and oxidized phospholipids were monitored by absorbance at 206 nm. The oxidation products were simultaneously monitored at 234 nm where the intact phospholipids have only a very slight end absorption. Second-derivative uv spectroscopy proved to be extremely useful to identify the presence or to verify the absence of oxidation products in phospholipid samples. For autoxidized docosahexaenoic acid containing phospholipids, the absorbance maximum of diene oxidation products is 237 nm for the trans,trans (t,t) isomer and 246 nm for the cis,trans (c,t) isomers. Similarly, five classes of triene oxidation product stereoisomers have distinct absorbance maxima detected by second-derivative spectroscopy ranging from 269 to 292 nm.

4-Hydroxyhexenal: a lipid peroxidation product derived from oxidized docosahexaenoic acid.

4-Hydroxynonenal and 4-hydroxyhexenal are cytotoxic aldehydic products of lipid peroxidation with high biological activity. Peroxidation of n - 6 fatty acids produces 4-hydroxynonenal, but the origin of 4-hydroxyhexenal has been uncertain. We now present evidence that 4-hydroxyhexenal is generated by oxidation of docosahexaenoic acid, the most abundant n-3 fatty acid in tissues.