Dye Release Experiments Research Articles

Synthesis of [Hexafluorovalyl1,1′]gramicidin S

Abstract [Hexafluorovalyl1,1′]gramicidin S (8), in which two valine residues in the natural gramicidin S were replaced by l-hexafluorovaline (Hfv) residues, was synthesized by the solid-phase-synthesis and cyclization-cleavage method on benzophenone oxime resin. Though the racemic hexafluorovaline derivative was employed for the peptide synthesis, the desired product was isolated in moderate yield, probably reflecting the stable cyclic structure of 8. CD and 1H NMR spectra indicated that the conformation of 8 is similar to that of gramicidin S. The two β-turn structure and antiparallel β-sheet structure with four intramolecular hydrogen bondings were maintained in spite of introducing the hexafluorovaline residues. The dye-release experiment from lecithin vesicle and antimacrobial activity assay also indicated that 8 showed membrane-disturbing activity like gramicidin S, although the activity of 8 was somewhat weaker than gramicidin S.

Conformational and functional study of magainin 2 in model membrane environments using the new approach of systematic double-D-amino acid replacement.

Systematic double-D-amino acid replacement of adjacent amino acids has been used to study the secondary structure of the amphiphilic, antibiotic peptide magainin 2 amide (M2a) by circular dichroism spectroscopy. Bound to liposomes, the secondary structure of the peptide is characterized by a weak alpha-helix in the N-terminus and a stable alpha-helix between residues 9 and 21. The lack of conformational differences in the peptide when bound to vesicles of varying negative charge density indicates marked independence of the structure from electrostatic forces. The similarity of the helicity profiles observed for double D-isomers bound to vesicles and in the presence of sodium dodecyl sulfate micelles (SDS) clearly shows that SDS can mimic magainin-lipid interactions. In contrast, in 1:1 trifluoroethanol/buffer (v/v), the peptide exhibits a weak alpha-helix extended from the N- to the C-terminus. Dye release experiments from vesicles of phosphatidylglycerol showed that double-D-amino acid substitution only in the region of the stable helix results in a reduction of the membrane-permeabilizing ability. On vesicles with a reduced amount of acidic phospholipids, double-D-amino acid substitution in any position leads to a drastic reduction of peptide-induced membrane permeabilization. Whereas the activity of M2a on phosphatidylglycerol was found to be mainly electrostatically determined, hydrophobic interactions play a decisive role in the interaction with vesicles of reduced negative charge density. Fluorescence investigations of tryptophan-containing analogs of high and low helicity showed that differences in the location of the chromophores of the membrane-bound peptides do not exist.

Two mode ion channels induced by interaction of acidic amphipathic α-helical peptides with lipid bilayers

In order to investigate the ion permeability and selectivity of ion channel formed by amphipathic α-helical peptides, we designed to synthesize an acidic peptide Ac(-Leu-Ala-Glu-Leu-) 3NHCH 3 (Glu-4 3) and its channel property was compared with a basic peptides Arg-4 3 in which Glu in Glu-4 3 was replaced by Arg. Two modes of the conductance change were observed by the interaction of Glu-4 3 with planar lipid bilayers; a steady increase of the conductance with the elapse of time (mode 1) and a spike-like increase of the current (mode 2) appearing with a lag time and overlapping the model current increase. The application of negative membrane potential induced the mode 1 current and the lowering pH decreased it, suggesting that the mode 1 current is caused by slow insertion of Glu-4 3 into the lipid bilayer and then by forming certain unknown bundles like semichannels. Mode 2 was found to be consisted of channel type opened-close current with several different conductances and relatively short opening lifetimes. There was no ion selectivity in the mode 1 current, whereas the mode 2 current was cation selective. The peptide Arg-4 3 has formed a cation-selective ion channel but not shown such two mode current changes. The membrane potential formation experiment in liposomes using DiSC 3(5) also showed the cation selectivity for Arg-4 3 and non-ion selectivity for Glu-4 3. The difference between Arg-4 3 and Glu-4 3 was also observed in conformational analysis by CD and in dye-release experiment from liposome. Such difference was discussed in terms of electrostatic interaction between peptides and lipid head groups.

Oceanic diffusion diagrams

Some empirical relations between diffusion characteristics are investigated by the use of carefully examined data from instantaneous dye-release experiments in the upper mixed layer of the sea. The data cover a time scale of diffusion ranging from 2 hr to 1 month and a length scale from 30 m to 100 km. Two kinds of ‘diffusion diagrams’ are prepared; one showing horizontal variance versus diffusion time and the other showing apparent diffusivity versus the scale of diffusion. The overall behaviors of the horizontal variance and of the apparent diffusivity are evidently different from those which the similarity theory of turbulence deduces. However, there still remains a possibility that the similarity theory may be valid locally with different values of the rate of turbulent energy dissipation. The diagrams provide a practical means to predict the rate of horizontal spread of substance from an instantaneous source.