A high-performance liquid chromatography-based fluorometric method for assaying serine hydroxymethyltransferase toward serine formation.
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.
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
Zhaoyang
Z
Wei
Sanja
S
Roje
D017360:Arabidopsis / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase; D005470:Fluorometry / Q000379:methods*; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D007700:Kinetics; D010069:Oxadiazoles / Q000737:chemistry; D012694:Serine / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry
C531567:4-fluoro-7-mercapto-2,1,3-benzoxadiazole; D010069:Oxadiazoles; D013438:Sulfhydryl Compounds; D012694:Serine; D012696:Glycine Hydroxymethyltransferase