DEPTH PROFILES OF PULMONARY SURFACTANT PROTEIN-B IN PHOSPHATIDYLCHOLINE BILAYERS, STUDIED BY FLUORESCENCE AND ELECTRON-SPIN-RESONANCE SPECTROSCOPY

Pulmonary surfactant-associated protein B (SP-B) has been isolated fro m porcine lungs and reconstituted in bilayers of dipalmitoylphosphatid ylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using differen t reconstitution protocols have been estimated from the changes induce d in the fluorescence emission spectrum of the single protein tryptoph an. All the different reconstituted SP-B-phospholipid preparations stu died had similar Kd values for the binding of the protein to the lipid s, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein f luorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 Angstrom, from the cente r of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as re sonance energy transfer from SP-B tryptophan to an acceptor probe loca ted in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on th e method of reconstitution. SP-B reconstituted from lipid/protein mixt ures in organic solvents is inserted more deeply in PC or DPPC bilayer s than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualit ative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label el ectron spin resonance (ESR) spectroscopy, and could represent differen t functional stages in the surfactant cycle.