1H-NMR of phosphatidylcholine liposomes at low p2H in the presence of a paramagnetic shift reagent.
1H-NMR spectra of egg phosphatidylcholine liposomes in 2H2O were obtained at several p2H values in the presence of 10 mM PrCl3 added after sonication of the phospholipid. It has been found that as the p2H is lowered below 2, the two distinct signals corresponding to the outer and inner phospholipid trimethylammonium groups which arise by the shifting effect of the paramagnetic cation on the external surface of vesicles, tend to coalesce into a single, high-field peak, at the position corresponding to the internal, non-shifted -N+ (CH3)3 protons. These results can be interpreted to mean that the shifting effect of Pr3+ on phosphatidylcholine NMR spectra, is due to electrostatic interaction between the lanthanide and the ionized group of the lipid. At low p2H, as the phosphodiester becomes protonated, the paramagnetic cation is no longer attracted by the liposome surface and its shifting effect on the phospholipid NMR signals disappears. The plot of the p2H dependence of the chemical shift of the outer trimethylammonium resonance of phosphatidylcholine liposomes with praseodymium ions present only on the outside of vesicles, results in a sigmoidal titration curve with its midpoint at p2H 1.5. In contrast, the inner signal is not affected by p2H. If coalescence of signals is considered as indicative of complete protonation of the phosphate moiety, the value of 1.5 can be taken as the apparent pK for the ionization of that group under the experimental conditions employed, i.e., 10 mM PrCl3 in 2H2O. That the low p2H-induced merging of the signals is reversible, is shown by the reappearance of the two peaks when the p2H of the phospholipid dispersion is raised from 1 to 5.7. Since the recovery of the trimethylammonium signal splitting indicates that Pr3+ has remained excluded from the liposome inner compartment, these experiments also demonstrate that the vesicles have not been disrupted by exposure to such an extreme acidic condition as p2H 1.
Fernández
M S
MS
Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del I.P.N., México D.F.
D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D010713:Phosphatidylcholines; D011221:Praseodymium
D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D003903:Deuterium; D011221:Praseodymium