Brugia Malayi - Clark Science Center - Smith College

A 39 K-NMR Study of the Encapsulation of Potassium in
Phosphatidylcholine Liposomes
Colby R. Loew
The objective of this project is to confirm the encapsulation of potassium cations in phosphatidylcholine liposomes though
implementation of 39 K-NMR. With working 39 K-NMR acquisition parameters established, distinct intra- and extra-liposomal
concentrations of potassium should be apparent upon the introduction of dysprosium as a shift reagent to the liposome sample.
The existence of two potassium environments as evidenced by 39 K-NMR will become paramount as the eventual goal is to
examine the cyclic peptide ionophore valinomycin for its ability to transport cations across the phospholipid bilayer.
As NMR was employed in this study, certain acquisition parameters proved to be imperative. The receiver gain is the degree
to which the signal coming from the sample is amplified and is much like the volume dial on a radio. When the receiver gain is
set too high, the FID becomes clipped which may lead to baseline distortions in the processed spectrum. A signal that is too
strong may also result in an “overflow” error of the receiver or analog to digital converter (ADC). Moreover a gain that is set too
low causes part of the sample signal to be lost. The pre-scan-delay time (DE) is the time in microseconds between the last pulse
and the beginning of data acquisition with the purpose of avoiding pulse feed through. If the pre-scan-delay time is not long
enough, a residual signal from the pulse may cause an overflow error. Through experimentation, values for these parameters were
determined so that 39 K-NMR data acquisitions were made using a DE value of 20 µsec and gain of 10.
The formation of vesicles is spontaneous upon hydration of a dry lipid film. In the presence of an aqueous solution,
phospholipids will spontaneously arrange so that the hydrophobic tails become isolated from the polar solvent using the
hydrophilic head groups as a barrier. These hydrophobic contacts are the principal interactions that promote the construction of
the lipid bilayer of the vesicle. Using a previously established extrusion protocol, phosphatidylcholine was hydrated in 200 mM
KCl to form liposomes. Upon introduction of dysprosium as a shift reagent, intra- and extra-liposomal potassium peaks have not
consistently become apparent in different sample preparations. In order to continue this study, the samples must be reliable in
producing two separate cation environments. As part of an honors project, I plan to continue this research project in hopes of
realizing the goal of using the potassium liposomes to study the transport abilities of the ionophore valinomycin. (Supported by
the Howard Hughes Medical Institute)
Advisor: Cristina Suarez
2012
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