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A nonradioactive assay for N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on o-phthaldialdehyde derivatization of methionine and fluorescence detection. 1991

A Garras, and R Djurhuus, and B Christensen, and J R Lillehaug, and P M Ueland
Department of Pharmacology and Toxicology, University of Bergen, Norway.

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.

UI MeSH Term Description Entries
D007202 Indicators and Reagents Substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. Indicators are substances that change in physical appearance, e.g., color, at or approaching the endpoint of a chemical titration, e.g., on the passage between acidity and alkalinity. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents. (From Grant & Hackh's Chemical Dictionary, 5th ed, p301, p499) Indicator,Reagent,Reagents,Indicators,Reagents and Indicators
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008099 Liver A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances. Livers
D008715 Methionine A sulfur-containing essential L-amino acid that is important in many body functions. L-Methionine,Liquimeth,Methionine, L-Isomer,Pedameth,L-Isomer Methionine,Methionine, L Isomer
D008809 Mice, Inbred C3H An inbred strain of mouse that is used as a general purpose strain in a wide variety of RESEARCH areas including CANCER; INFECTIOUS DISEASES; sensorineural, and cardiovascular biology research. Mice, C3H,Mouse, C3H,Mouse, Inbred C3H,C3H Mice,C3H Mice, Inbred,C3H Mouse,C3H Mouse, Inbred,Inbred C3H Mice,Inbred C3H Mouse
D009764 o-Phthalaldehyde A reagent that forms fluorescent conjugation products with primary amines. It is used for the detection of many biogenic amines, peptides, and proteins in nanogram quantities in body fluids. ortho-Phthalaldehyde,Orthophthaldialdehyde,o-Phthaldialdehyde,ortho-Phthalic Aldehyde,Aldehyde, ortho-Phthalic,o Phthalaldehyde,o Phthaldialdehyde,ortho Phthalaldehyde,ortho Phthalic Aldehyde
D011919 Rats, Inbred Strains Genetically identical individuals developed from brother and sister matings which have been carried out for twenty or more generations or by parent x offspring matings carried out with certain restrictions. This also includes animals with a long history of closed colony breeding. August Rats,Inbred Rat Strains,Inbred Strain of Rat,Inbred Strain of Rats,Inbred Strains of Rats,Rat, Inbred Strain,August Rat,Inbred Rat Strain,Inbred Strain Rat,Inbred Strain Rats,Inbred Strains Rat,Inbred Strains Rats,Rat Inbred Strain,Rat Inbred Strains,Rat Strain, Inbred,Rat Strains, Inbred,Rat, August,Rat, Inbred Strains,Rats Inbred Strain,Rats Inbred Strains,Rats, August,Rats, Inbred Strain,Strain Rat, Inbred,Strain Rats, Inbred,Strain, Inbred Rat,Strains, Inbred Rat
D002461 Cell Line, Transformed Eukaryotic cell line obtained in a quiescent or stationary phase which undergoes conversion to a state of unregulated growth in culture, resembling an in vitro tumor. It occurs spontaneously or through interaction with viruses, oncogenes, radiation, or drugs/chemicals. Transformed Cell Line,Cell Lines, Transformed,Transformed Cell Lines
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man

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