LIPID EFFECTS ON AGGREGATION OF PULMONARY SURFACTANT PROTEIN SP-C STUDIED BY FLUORESCENCE ENERGY-TRANSFER

The self-association of pulmonary surfactant protein SP-C in lipid ves icles was studied using fluorescence energy transfer. Bovine SP-C was labeled with two fluorescent probes, succinimidyl (7-nitrobenz-2-oxa-1 ,3-diazol-4-yl)amino]hexanoate and eosin isothiocyanate, on the amino terminus of the protein, producing NBD-SP-C and EITC-SP-C, respectivel y. The N-terminus of SP-C was relatively immobile between 20 and 37-de grees-C, as demonstrated by high fluorescence anisotropy of NBD-SP-C a nd EITC-SP-C. The mobility increased at the transition of the lipid to the fluid phase. Using fluorescence energy transer, with NBD-SP-C as the donor and EITC-SP-C as the acceptor, a high degree of SP-C/SP-C as sociation was found below 25-degrees-C, decreasing to very little self -association above 42-degrees-C in 7:1 hatidylcholine-1,2-dipalmitoylp hosphatidylglycerol (DPPC-DPPG) vesicles. The fraction of SP-C aggrega ted below 37-degrees-C in 7:1 DPPC-DPPG was estimated from the observe d energy transfer to be more than 70% of total SP-C. In various lipid mixtures, self-association of SP-C was dependent on the presence of at least some gel-phase lipids. In a lipid mixture resembling pulmonary surfactant, gradually increasing self-association was observed below 3 8-degrees-C. The relation of the present data to the state of aggregat ion of SP-C in pulmonary surfactant is discussed.