IDENTIFICATION OF SELENOCYSTEINE AND SELENOMETHIONINE IN PROTEIN HYDROLYSATES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF THEIR O-PHTHALDIALDEHYDE DERIVATIVES
C. Hammel et al., IDENTIFICATION OF SELENOCYSTEINE AND SELENOMETHIONINE IN PROTEIN HYDROLYSATES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF THEIR O-PHTHALDIALDEHYDE DERIVATIVES, Analyst, 122(11), 1997, pp. 1359-1363
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 deco
mposition of selenocysteine during this process, After precolumn deriv
atization of the amino acids with o-phthaldialdehyde, the hydrolysate
was chromatographed on C-18 columns. The selenoamino acids were detect
ed either by the fluorescence of their o-phthaldialdehyde derivatives
(detection limit 30 pmol for selenomethionine and 170 pmol for selenoc
ysteine) or by selenium determination in the eluate using atomic absor
ption spectrometry (detection limit 0.3 pmol) or, with Se-75-labelled
compounds, the measurement of the tracer activity. With the latter pro
cedure the detection limit, which depends on the specific activity of
the Se tracer, could be decreased to the femtomole range. The method w
as successfully applied to the identification of selenocysteine in sev
eral newly found mammalian selenium-containing proteins.