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 surfactant proteins (SP)-B and SP-C were studied for separation efficiency and surface activity of the isolated proteins recombined with synthetic phospholipids (SPL). SP-B and SP-C were obtained from calf lung surfactant extract by C8 chromatography with isocratic elution by either of three solvent systems: 7:1:0.4 MeOH/CHCl(3)/5% 0.1 M HCl (solvent A), 7:1 MeOH/CHCl(3)+ 0.1% TFA (solvent B), and 7:1:0.4 MeOH/CHCl(3)/H(2)O + 0.1% TFA (solvent C). Solvents A and C yielded pure apoprotein in a single pass, with estimated total protein recoveries of >85 and >90%, respectively. Solvent B was less effective in purifying 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 with 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 milligrams of virtually lipid-free SP-B and SP-C in a single column pass will facilitate research on their biological, structural, and biophysical properties.
Baatz
J E
JE
Department of Pediatrics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-3313, USA. baatzje@musc.edu
Zou
Y
Y
Cox
J T
JT
Wang
Z
Z
Notter
R H
RH
D000327:Adsorption / Q000187:drug effects; D000818:Animals; D002417:Cattle; D002845:Chromatography; D010743:Phospholipids / Q000378:metabolism; D011510:Proteolipids / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D011663:Pulmonary Surfactants / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D020539:Sequence Analysis, Protein; D012997:Solvents / Q000494:pharmacology; D013500:Surface Tension / Q000187:drug effects
D010743:Phospholipids; D011510:Proteolipids; D011663:Pulmonary Surfactants; D012997:Solvents