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 chlo...

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Bibliographic Details
Published in:Protein expression and purification 2001-11, Vol.23 (2), p.319-327
Main Authors: Bünger, Harald, Krüger, Ralph-Peter, Pietschmann, Sylvia, Wüstneck, Nadeshda, Kaufner, Lutz, Tschiersch, Renate, Pison, Ulrich
Format: Article
Language:English
Subjects:
Animals
captive bubble
Chromatography, High Pressure Liquid - methods
MALDI-TOF-MS
preparative HPLC
Proteolipids - analysis
Proteolipids - chemistry
Pulmonary Surfactants - analysis
Pulmonary Surfactants - chemistry
Sheep
SP-B
SP-C
surface pressure
surfactant proteins
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Summary: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.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2001.1510