Incorporation of hydrophilic protein modified with hydrophobic agent into liposome membrane.

The hydrophilic protein-enzyme, alpha-chymotrypsin, can be bound to the liposomal membrane after the preliminary increase in hydrophobicity induced by acylation of protein amino groups with palmitic chloroanhydride. The efficacy of binding depends on the degree of modification. The bound enzyme almost completely preserves its catalytic properties and the ability to interact with a high molecular weight inhibitor. Binding can be performed during both the process of liposome formation and the incubation of a modified enzyme with preformed liposomes. According to ESR and fluorescence spectroscopy, the hydrophobic tail of the modified enzyme is incorporated into the membrane and the protein globule is located on the surface of the membrane. Protein incorportation causes an increase in the amorphous nature of the membrane, and the bound protein is not as mobile as the free protein. The approach discussed can be useful in binding soluble hydrophilic proteins to artificial membranes.

FITC-labeled lipopolysaccharide: use as a probe for liposomal membrane incorporation studies.

FITC-labeled LPS from Neisseria meningitidis can be used as a probe to follow the process of LPS incorporation into liposomal membrane and to study its interaction with a bilayer. The incorporation of FITC-LPS into the bilayer was proved by physicochemical methods as well as by liposomal LPS toxicity decrease in actinomycin D-sensitized mice. Fluorescence intensity increase was observed upon the insertion of FITC-LPS into the membrane of dehydration/rehydration vesicles and vesicles obtained by co-sonication of lipid suspension and FITC-LPS. Following FITC-LPS fluorescence polarization it was shown that the substance seems to be clusterized in the liposomal membrane starting from FITC-LPS/lipid molar ratio 1:800.

Free radical label: new approach to the study of super-slow molecular dynamics of lipid systems.

A new method of EPR-spectroscopy, the recombination of free radicals appearing as a result of indirect radiolysis of biological molecules after a low temperature irradiation, was applied to the study of molecular dynamics of dimyristoyl phosphatidylcholine in mass and in the structure of liposomes above and below the transition temperature. It was shown that the mobility of lipid molecules in crystalline liposomes was lower than in the structure of liquid-crystalline liposomes. The addition of cholesterol in liposome membranes decreased the lateral molecular motion of lipids in crystalline and liquid-crystalline states; in the latter case, the effect of cholesterol addition was more pronounced. The activation energy for the displacement of the fragments of lipid molecules and the lipid molecule as a whole was estimated, and it was shown that the lipid matrix possesses a high degree of heterogeneity. The solubility of oxygen in the lipid bilayer and the mechanism of lipid diffusion are discussed.