Determination of proteins, phosphatidylethanolamine, and phosphatidylserine in organic solvent extracts of tissue material by analysis of phenylthiocarbamyl derivatives.

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.