cytochrome-c-t and dimyristoylphosphatidylglycerol

Unilamellar liposomes, prepared from synthetic lipid mixture of DMPC and DMPG either by sonication or extrusion, were used to entrap water soluble and water insoluble molecules to investigate the efficacy of encapsulation by different liposome preparation methods. In the case of entrapment of hydrophilic protein cytochrome-C, the solutions were subjected to a series of ultrafiltration steps to eliminate any free protein outside the vesicles. It was observed that the protein could be encapsulated by the vesicles only if cholesterol was present in the bilayer. The release of cytochrome-C was observed spectrophotometrically upon vesicle-breakdown. The amount of protein encapsulated depended on the method of preparation and was found to be 10 times greater in extruded liposomes compared to those produced by sonication. Hydrophobic Vitamin E, on the other hand, could be encapsulated in the liposome bilayer, independently of the presence of cholesterol and the method of preparation. These fundamental results can be used to develop more efficient drug encapsulations and to have better understanding about their release.

Topics: Chemistry, Pharmaceutical; Cholesterol; Cytochromes c; Dimyristoylphosphatidylcholine; Drug Compounding; Hydrophobic and Hydrophilic Interactions; Liposomes; Particle Size; Phosphatidylglycerols; Sonication; Ultrafiltration; Vitamin E

Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c-lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.

Topics: Cytochromes c; Fractionation, Field Flow; Hydrogen-Ion Concentration; Lipids; Oleic Acid; Particle Size; Phosphatidylglycerols; Proteins; Sodium Dodecyl Sulfate; Static Electricity