QUANTITATIVE FLUORESCENCE ANALYSIS OF THE ADSORPTION OF LYSOZYME TO PHOSPHOLIPID-VESICLES

Experiments directed to measure the interaction of lysozyme with lipos omes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS ) have been conducted by monitoring both protein and lipid fluorescenc e and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of ana lysis in which the fractional contribution at moderate binding conditi ons is determined from either total fluorescence decay or anisotropy d ecay curves of tryptophan at limiting binding conditions. In the energ y transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy accepters of the excited tryptophan residues i n lysozyme. The binding was strongly dependent on the molar fraction o f negatively charged PS in neutral PC membranes and on the ionic stren gth. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conform ational rearrangements induced by binding of the protein to these lipi d membranes. The dynamics of membrane bound protein appeared to be dep endent on the physical state of the membrane. Independent of protein f luorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction o f lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/P C membranes resulted in a substantial decrease of the intramolecular e xcimer formation, while the excimer formation of dipyrenyl-labelled ph osphatidylcholine in neutral PC membranes barely changed in the presen ce of lysozyme.