High-performance liquid chromatography of phenylthiocarbamyl-amino acids. Application to carboxyl-terminal sequencing of proteins.
A carboxyl-terminus microsequencing method has been developed on the basis of carboxypeptidase A digestion and derivatization of released amino acids with phenylisocyanate followed by subsequent high-performance liquid chromatographic analysis of the resulting phenylthiocarbamyl amino acids. This technique has been used to confirm the carboxyl-terminal sequence of bovine brain-derived acidic fibroblast growth factor. The method is of general application for the microsequencing of the carboxyl-termini of proteins. Since identification of derivatized amino acids was performed on the entire reaction mixture following digestion, a new chromatographic identification procedure for phenylthiocarbamyl amino acids was developed in order to resolve them from spurious peaks. This procedure can be applicable to amino acid analysis.
Gimenez-Gallego
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Department of Biochemistry and Molecular Biology, Merck Institute for Therapeutic Research, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065.
Thomas
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KA
D000596:Amino Acids / Q000032:analysis*; D002268:Carboxypeptidases; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D010670:Phenylthiourea / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D014357:Trypsin
D000596:Amino Acids; D011506:Proteins; D010670:Phenylthiourea; D002268:Carboxypeptidases; D014357:Trypsin