title: Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient1H NMRin situ.
abstract: The kinetics of binding, the diffusivity, and the binding amount of a neuropeptide, leucine-enkephalin (L-Enk) to lipid bilayer membranes are quantified by pulsed-field-gradient (PFG)1H NMRin situ. The peptide signal is analyzed by the solution of the Bloch equation with exchange terms in the presence of large unilamellar vesicles (LUVs) as confined, but fluid model cell membranes. Even in the case that the membrane-bound and the free states of L-Enk cannot be distinguished in the one-dimensional NMR spectrum, the PFG technique unveils the bound component of L-Enk after the preferential decay of the free component at the high field gradient. In 100-nm diameter LUVs consisting of egg phosphatidylcholine, the rate constants of the peptide binding and dissociation are 0.040 and 0.40 s-1at 303 K. This means that the lifetime of the peptide binding is of the order from second to ten-second. The diffusivity of the bound L-Enk is 5×10-12m2/s, almost 60 times as restricted as the movement of free L-Enk at 303K. One-tenth of 5mM L-Enk is bound to 40mM LUV. The binding free energy is calculated to be -2.9 kJ/mol, the magnitude close to the thermal fluctuation, 2.5 kJ/mol. The result demonstrates the potential of PFG1H NMR to quantify molecular dynamics of the peptide binding to membranes.
authors.affiliation: Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, 7-2-1, Kamiohno, Himeji 670-8524, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yoshii
authors.affiliation: Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, 7-2-1, Kamiohno, Himeji 670-8524, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Emoto
authors.affiliation: Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, 7-2-1, Kamiohno, Himeji 670-8524, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Okamura
keywords: drug delivery; exchange; fluid membrane dynamics; free energy of transfer from water to membrane; mobility