Differential extraction for the rapid purification of bovine surfactant protein B.
Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.
Beers
M F
MF
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia.
Bates
S R
SR
Fisher
A B
AB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D008168:Lung / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011510:Proteolipids / Q000302:isolation & purification*; D011663:Pulmonary Surfactants / Q000302:isolation & purification*; D012997:Solvents; D014465:Ultrasonics
D000911:Antibodies, Monoclonal; D010455:Peptides; D011510:Proteolipids; D011663:Pulmonary Surfactants; D012997:Solvents