Identification of selenocysteine and selenomethionine in protein hydrolysates by high-performance liquid chromatography of their o-phthaldialdehyde derivatives

A method for the identification of selenocysteine and selenomethionine in protein hydrolysates was developed, The proteins were subjected to acid hydrolysis after they had been carboxymethylated to prevent decomposition of selenocysteine during this process, After precolumn derivatization of the amino acids with o-phthaldialdehyde, the hydrolysate was chromatographed on C-18 columns. The selenoamino acids were detected either by the fluorescence of their o-phthaldialdehyde derivatives (detection limit 30 pmol for selenomethionine and 170 pmol for selenocysteine) or by selenium determination in the eluate using atomic absorption spectrometry (detection limit 0.3 pmol) or, with Se-75-labelled compounds, the measurement of the tracer activity. With the latter procedure the detection limit, which depends on the specific activity of the Se tracer, could be decreased to the femtomole range. The method was successfully applied to the identification of selenocysteine in several newly found mammalian selenium-containing proteins.