TY - JOUR A1 - Bünger, H. A1 - Krüger, Ralph-Peter A1 - Pietschmann, S. A1 - Wüstneck, N. A1 - Kaufner, L. A1 - Tschiersch, R. A1 - Pison, U. T1 - Two hydrophobic protein fractions of ovine pulmonary surfactant: Isolation, characterisation and biophysical activity JF - Protein expression and purification N2 - 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. KW - Surfactant proteins KW - SP-B KW - SP-C KW - Preparative HPLC KW - MALDI-TOF-MS KW - Surface pressure KW - Captive bubble PY - 2001 DO - https://doi.org/10.1006/prep.2001.1510 SN - 1046-5928 SN - 1096-0279 VL - 23 IS - 2 SP - 319 EP - 327 PB - Elsevier CY - Amsterdam AN - OPUS4-2174 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -