A quantitative method for detecting surfactant-associated protein C in pulmonary surfactant.
We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated 3.5-kDa protein, based on the release of free-sulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with [14C]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with 1% KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the 14C-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels (approximately 100 ng) of SP-C and is not affected by surfactant protein B or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids (approximately 30% at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein.
Qanbar
R
R
Department of Biochemistry, University of Western Ontario, London, Canada.
Possmayer
F
F
D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D002845:Chromatography / Q000379:methods; D005021:Ethylamines; D007460:Iodoacetamide; D009185:Myelin Proteins / Q000032:analysis; D011510:Proteolipids / Q000032:analysis* / Q000302:isolation & purification; D011663:Pulmonary Surfactants / Q000032:analysis* / Q000302:isolation & purification; D011817:Rabbits; D011869:Radioligand Assay; D012015:Reference Standards; D012680:Sensitivity and Specificity
D002250:Carbon Radioisotopes; D005021:Ethylamines; D009185:Myelin Proteins; D011510:Proteolipids; D011663:Pulmonary Surfactants; C016162:triethylamine; D007460:Iodoacetamide