A nuclear-magnetic-resonance-based assay for betaine-homocysteine methyltransferase activity

Betaine-homocysteine methyltransferase (BHMT) activity can be measured directly and kinetically by (1)H-nuclear magnetic resonance spectroscopy. The disappearance of substrates and the formation of products are monitored simultaneously. Alternative substrates, separately and when mixed with glycine betaine, can also be monitored. Each assay can be completed in 1h. Using 2mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH 7.5) and measuring the production of N,N-dimethylglycine, the CV is 6.3% (n=6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed, by measuring the N,N-dimethylglycine produced after incubation with 2 mM glycine betaine and homocysteine (CV=5.3%, n = 6) with a detection limit of 2 nkatal. These assays were used to show that the natural betaines trigonelline, proline betaine, arsenobetaine, and l-carnitine are neither substrates nor significant inhibitors of rat liver BHMT, that the thetins dimethylthetin and dimethylsulfoniopropionate are substrates and inhibit methyl transfer from glycine betaine, and that the K(m) for glycine betaine is 0.19+/-0.03 mM with a V(max) of 17+/-0.7 nMol min(-1) mg(-1).