Isocratic resolution of amino acid thiohydantoins by high-performance liquid chromatography.

Sixteen amino acid thiohydantoins encountered during COOH-terminal degradation of peptides and proteins with ammonium thiocyanate have been separated and identified by reverse phase high-performance liquid chromatography using a DuPont Zorbax ODS column and a novel procedure for preparation of thiohydantoin derivative of serine and threonine is described. Three isocratic systems were employed: System 1, buffer A alone (0.01 n sodium acetate, pH 4.5); system 2, 10% acetonitrile in buffer A; system 3, 24% acetonitrile in buffer A. All 16 thiohydantoins could be resolved using a combination of systems 1, 2, and 3. The method is rapid and sensitive (1 nmol) and constitutes a step toward automation of the solid-phase COOH-terminal sequencing method.