Fractionation of soybean phospholipids by preparative high-performance liquid chromatography with sorbents of various particle size.
Normal-phase high-performance liquid chromatography was used on a preparative scale to seperate phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) phospholipids from soybean. Separation was achieved using mixtures of three solvents, hexane, methanol and isopropanol. The optimized mobile phase compositions were experimentally determined while operating in a linear gradient mode using 15, 5-20, 25-40, and 40-63 microm preparative particles as well as 4 microm analytical particles. A gradient mobile phase was established on a commmercially available analytical Nova-Pak column such that hexane linearly decreased from 85 to 0 as isopropanol and methanol linearly increased in two gradient steps from 10 to 30 and 5 to 70 respectively. The total run time was 25 min at a flow-rate of 1.5 ml/min. A slight change in mobile phase composition was required to increase the resolution of phospholipids. The 15 microm particle size gave the best separation of the preparative particle sizes examined based on their resolutions between PE and PI and PI and PC. Finally, the retention factors of PE and PC were correlated in terms of mobile phase composition.
Development of oxidation models and analytical techniques specific to phospholipids and their degradation products in functional foods
Among the numerous beneficial effects of phospholipides can highlight their role in protecting the cardiovascular system and improving memory and learning. These effects can be partially explained by the fact that phospholipids are rich in polyunsaturated (eicosapentaenoic and docosahexaenoic acids) or essential fatty acids. Thus, it is very significant to explore potential food sources containing polyunsaturated rich phospholipids for the development new food products supplemented with phospholipids.This work allowed a series method for the extraction, purification, separation and quantification of phospholipids in order to characterize the polyunsaturated phospholipid sources. Food supplementation with polyunsaturated phospholipids rises however the question of their stability during food processing at high temperature. Then we have developed methods to identify phospholipids degradation products and determine their kinetics of the oxidation during thermal treatment which is a widespread food processing.Phospholipids (PE and PC) are more stable than triacylglyceride, this is due to the presence of an amino group in the molecule.
Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers.
Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.