A nonradioactive assay for N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on o-phthaldialdehyde derivatization of methionine and fluorescence detection.
The enzyme N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase, EC 2.1.1.13) catalyzes the conversion of homocysteine to methionine in the presence of a reducing system. N5-Methyltetrahydrofolate serves as a methyl donor in this reaction. An assay for the enzyme is described, which is based on methionine quantitation by o-phthaldialdehyde (OPA) derivatization and reversed-phase liquid chromatography. The enzymatic reaction is linear for at least 120 min under reducing conditions (125 mM 2-mercaptoethanol) and running the assay below an oil layer. This reducing system does not interfere with formation of the methionine-OPA adduct, which is separated from interfering compounds and an internal standard (norvaline) by a mobile phase adjusted to pH 5.0. The inclusion of internal standard increases the precision of the assay and corrects for the variable fluorescence yield due to occasional inaccurate pH adjustment before the derivatization step. Norvaline was suitable for this purpose because it elutes close to methionine and is not a natural amino acid present in biological extracts. This nonradioactive assay for methionine synthase was evaluated by comparison with a conventional method based on isolation of radioactive methionine by anion-exchange chromatography and by determination of enzyme activity in extract from cultured cells and liver.
Garras
A
A
Department of Pharmacology and Toxicology, University of Bergen, Norway.
Djurhuus
R
R
Christensen
B
B
Lillehaug
J R
JR
Ueland
P M
PM
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001322:Autoanalysis / Q000379:methods; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008099:Liver / Q000201:enzymology*; D008715:Methionine / Q000032:analysis*; D051379:Mice; D008809:Mice, Inbred C3H; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods; D009764:o-Phthalaldehyde
D007202:Indicators and Reagents; D009764:o-Phthalaldehyde; D008715:Methionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase