The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.
Dediazoniation of 2,4,6-trimethylbenzenediazonium tetrafluoroborate, 1-ArN 2 BF 4 (for the z-Ar compounds described in this paper, z refers to the length of the carbon chain of the substituent at C4 of the benzene ring), in aqueous solutions containing sodium methyl sulfate, NaMeSO 4 , or sodium methanesulfonate, NaMeSO 3 , yields 2,4,6-trimethylphenol, 1-ArOH, 2,4,6-trimethylphenyl methyl sulfate, 1-ArOSO 3 Me and 2,4,6-trimethylphenyl methanesulfonate, 1-ArO 3 SMe, respectively. The relative yields of 1-ArO 3 SMe or 1-ArOSO 3 Me and 1-ArOH depend on the NaMeSO 4 or NaMeSO 3 concentrations. 4-n-Hexadecyl-2,6-dimethylbenzenediazonium tetrafluoroborate, 16-ArN 2 BF 4 , was used to determine the local head group concentration in sodium dodecyl sulfate and sodium dodecanesulfonate micelles by chemical trapping comparing the relative product yields with those obtained in water using the short chain analogs.Ab initio calculations of the spontaneous dediazoniation of phenyldiazonium ion in the gas phase, as well as in aqueous solution with, or without, added MeSO 3 Ϫ , yield potential energy surfaces for the reaction. For this model the calculated and experimental values of the spontaneous dediazoniation rate constants in aqueous solution, as well as the product composition, were similar to those obtained with 1-ArN 2 ϩ . These results suggest that in aqueous solution nucleophiles can only compete with water if a diazonium ionؒnucleophile complex is formed prior to N 2 loss. Calculations show that the addition of nucleophiles to the arenediazonium ion occurs without a saddle point in the potential energy surface, suggesting that the free phenyl cation is not an obligatory intermediate in aqueous solutions.
The increasing demand for fatty acid-free lecithin required modifications in existing purification methods. In this technical note we describe a purification procedure with the following steps: a) homogenization and extraction of yolks obtained from fresh eggs with acetone, b) solubilization with ethanol and solvent elimination and c) repeated solubilization/precipitation with petroleum ether/acetone. This crude extract was chromatographed on neutral alumina, which was exhaustively washed with chloroform before elution with chloroform:methanol, allowing the sequential separation of fatty acids and lecithin. Chromatographic behavior and mass spectra of the product are presented. This fast procedure yields fatty acid-free lecithin at a competitive cost
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