High-yield purification of lung surfactant proteins SP-B and SP-C and the effects on surface activity

Several protocols for purification of milligram quantities of lung surfacta nt proteins (SP)-B and SP-C were studied for separation efficiency and surf ace activity of the isolated proteins recombined with synthetic phospholipi ds (SPL). SP-B and SP-C were obtained from calf lung surfactant extract by CS chromatography with isocratic elution by either of three solvent systems : 7:1:0.4 MeOH/CHCl3/5% 0.1 M HCl (solvent A), 7:1 MeOH/CHCl3+ 0.1% TFA (so lvent B), and 7:1:0.4 MeOH/CHCl3/H2O + 0.1% TFA (solvent Q. Solvents A and C yielded pure apoprotein in a single pass, with estimated total protein re coveries of > 85 and > 90%, respectively. Solvent B was less effective in p urifying SP-B and SP-C, had a lower recovery efficiency, and gave isolates with less surface activity. Mixtures of SPL plus SP-B eluted with solvents A and C adsorbed to equilibrium surface tensions of 21-22 mN/m and reached minimum surface tensions <1 mN/m during dynamic cycling. Mixtures of SPL wi th SP-C obtained with solvents A and C had equilibrium surface tensions of 26-27 mN/m and minimum dynamic values of 2-7 mN/m. The ability to obtain mi lligrams of virtually lipid-free SP-B and SP-C in a single column pass will facilitate research on their biological, structural, and biophysical prope rties. (C) 2001 Academic Press.