A high-performance liquid chromatography-based fluorometric method for assaying serine hydroxymethyltransferase toward serine formation.

ABSTRACT

A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H(4)PteGlu(n) has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD-serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD-serine is quantified by comparison with standards. This method was used to determine the K(m) and k(cat) values for 5,10-CH(2)-H(4)PteGlu(5) of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH(2)-H(4)PteGlu(5) for an SHMT from any organism.