Two hydrophobic protein fractions of ovine pulmonary surfactant: isolation, characterization, and biophysical activity.
Pulmonary surfactant contains two extremely hydrophobic proteins, SP-B and SP-C. We present a novel HPLC method for the preparation of these hydrophobic proteins. It is based on size-exclusion chromatography using the apolar stationary-phase butyl silica gel and isocratic elution with acidified chloroform/methanol. Samples for HPLC were prepared from sheep lung lavage fluid by centrifugation and extraction with chloroform/methanol. Amino acid analyses of the two protein fractions revealed sequences that are consistent with SP-B and SP-C, respectively. MALDI-TOF-MS analyses of the SP-B fraction showed one major peak of dimeric SP-B with m/z 17,361, and additional peaks of monomeric and oligomeric forms, which are predominantly even numbered. The SP-C fraction showed a peak at m/z 4200, consistent with the theoretical mass of the dipalmitoylated form of this protein. The biophysical activity of pure sheep SP-B and SP-C was evaluated by measuring the surface tension using axisymmetric drop shape analysis for captive bubbles. We found distinct surface pressure versus surface area isotherms of SP-B and SP-C indicating different biophysical activities for these surfactant proteins. The new preparative HPLC method is able to replace the established, time-consuming low-pressure liquid chromatography method for the isolation of SP-B and SP-C from lipids.
Bünger
H
H
Klinik für Anästhesiologie und operative Intensivmedizin, Charité, Campus Virchow-Klinikum, Medizinische Fakultät der Humboldt-Universität zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.
Krüger
R P
RP
Pietschmann
S
S
Wüstneck
N
N
Kaufner
L
L
Tschiersch
R
R
Pison
U
U
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D011510:Proteolipids / Q000032:analysis / Q000737:chemistry*; D011663:Pulmonary Surfactants / Q000032:analysis / Q000737:chemistry*; D012756:Sheep
D011510:Proteolipids; D011663:Pulmonary Surfactants