Use of High-Pressure Liquid Chromatography with Fluorescence Detection for the in vitro Assay of S-Carboxymethyl-L-Cysteine S-Oxygenase

Abstract

The analysis of S-carboxymethyl-L-cysteine S-oxygenase activity by descending paper chromatography and chloroplatinate visualization is a cost effective but time consuming analytical procedure with 20 enzyme assays taking 88 h to analyse. Here we report on faster and more sensitive method of analysing S-carboxymethyl-L-cysteine S-oxygenase activity using high-pressure liquid chromatography with fluorescence detection following pre-column derivatization with o-phthaldehyde/2-mercaptoethanol. A base line separation of both S-carboxymethyl-L-cysteine R and S S-oxides was achieved using a 5μ, 30 × 2.1 mm id Hypersil Amino Acid C₁₈ column with a gradient elution. The method was linear over the concentration range 2.5–250 μM for both S-carboxymethyl-L-cysteine R and S S-oxides. The total analysis time for the 20 enzyme assays was reduced from 88 h to11 h 40 min.