Characterization of nonradioactive assays for cobalamin-dependent and cobalamin-independent methionine synthase enzymes.
Methionine synthase enzymes catalyze methyl group transfer from 5-methyltetrahydrofolate to homocysteine to give methionine and tetrahydrofolate. Assays for this enzyme activity usually monitor transfer of a 14C-methyl group from the N5-position of methyltetrahydrofolate to homocysteine to produce 14C-methionine that must be purified by anion-exchange chromatography. Alternatively, tetrahydrofolate may be derivatized with a formylating agent under acidic conditions to produce methenyltetrahydrofolate. We report optimization of this reaction for assay of cobalamin-dependent methionine synthase to give an economical method for determining enzyme activity that does not require the use of radioactive compounds. By heating for 10 min in 1 N hydrochloric acid containing 12% formic acid, the enzymatic product tetrahydrofolate is converted into methenyltetrahydrofolate, which absorbs light at 350 nm, while residual substrate 5-methyltetrahydrofolate does not contribute to the absorbance at 350 nm. The assay allows the derivatized product to be characterized in situ with a minimal increase in volume upon acidification. The results of the spectrophotometric assay given here have been compared with the radioactive assay to confirm the validity of the derivatization under the assay conditions. We also report the extension of this assay method for use in activity measurements of cobalamin-independent methionine synthase.
Drummond
J T
JT
Biophysics Research Division, University of Michigan, Ann Arbor 48109-1055, USA.
Jarrett
J
J
González
J C
JC
Huang
S
S
Matthews
R G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000032:analysis*; D000818:Animals; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology; D011868:Radioisotopes; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014805:Vitamin B 12 / Q000737:chemistry*
D007527:Isoenzymes; D011868:Radioisotopes; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12