title: Determination of proteins, phosphatidylethanolamine, and phosphatidylserine in organic solvent extracts of tissue material by analysis of phenylthiocarbamyl derivatives.
abstract: Amino acid analysis of organic solvent extracts of tissue material has been evaluated for determination of protein content. Conventional ninhydrin-based analysis does not allow determination of a large number of lipid-rich samples. Therefore, the hydrolyzed samples were treated with phenylisothiocyanate and the phenylthiocarbamyl (PTC) derivatives obtained were separated by reverse-phase HPLC. With this method, analysis of many lipid-rich samples is feasible. In addition, phosphatidylethanolamine and phosphatidylserine can then be determined together with the amino acid constituents. The PTC/reverse-phase HPLC method was used for analysis of chloroform/methanol extracts of spinal cord, lung, and bile after chromatography on Lipidex 5000 in methanol/ethylene chloride, 4:1 (v/v). The chromatography profiles show that in all tissue samples the proteins elute before the phospholipids. Consequently, a single step of Lipidex 5000 chromatography can be used to purify polypeptides present in organic solvent extracts. Using pulmonary surfactant extracts (with about 98% phospholipids and 1-2% proteins), we find that individual contents of surfactant proteins B and C can be determined by amino acid analysis.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Stark
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: O
authors.initials: O
authors.lastname: Danielsson
authors.forename: H
authors.initials: H
authors.lastname: Jörnvall
authors.forename: J
authors.initials: J
authors.lastname: Johansson
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D008168:Lung / Q000737:chemistry; D010670:Phenylthiourea / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000032:analysis*; D010718:Phosphatidylserines / Q000032:analysis*; D011510:Proteolipids / Q000032:analysis*; D011663:Pulmonary Surfactants / Q000032:analysis*; D012997:Solvents; D013116:Spinal Cord / Q000737:chemistry; D013552:Swine
chemical_list: D000596:Amino Acids; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011510:Proteolipids; D011663:Pulmonary Surfactants; D012997:Solvents; D010670:Phenylthiourea