Determination of the pKa of membrane-bound N,N-dimethylsphingosine using deuterium NMR spectroscopy

  • Lau B
  • Macdonald P
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Abstract

Deuterium nuclear magnetic resonance (2H-NMR) spectroscopy was applied to determine the pKaof the protein kinase C (PKC) inhibitor, N,N-dimethylsphingosine (DMS), when bound to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The quadrupolar splittings from the deuterium labels at the α-and the β-choline positions of the headgroup of POPC responded to the presence of DMS in a manner indicative of an accumulation of cationic charges near the surface plane occupied by the phospholipid phosphate group. Both quadrupolar splittings varied linearly with the amount of added DMS at pH 7.0. Conversely, at pH 10.0 DMS had virtually no influence on either quadrupole splitting, an effect attributed to titration of the dimethyl amino group of DMS to its neutral form. A DMS titration curve was obtained by quantifying the change in the quadrupolar splittings as a function of pH. The pKaof membrane-bound DMS was extracted from this2H-NMR data by simulating the quadrupole splitting-titration curve for different values of the pKa, yielding a pKaof 8.8 after non-linear least squares fitting. © 1995.

Author-supplied keywords

  • 2H-
  • Membrane-bound pKa, pKavalue
  • N,N-Dimethylsphingosine
  • NMR

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Authors

  • Bienca Lau

  • Peter M. Macdonald

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