5-Methyltetrahydrofolic acid disodium salt

5-Methyltetrahydrofolic acid disodium salt (CAS# 68792-52-9) is a folate derivative and the sodium salt form of N-Methyltetrahydrofolic acid (M338145).

5-Methyl-THF acts as both cofactor and substrate in the regeneration of methionine from homocysteine through the action of 5-methyl-THF-homocysteine S-methyltransferase.

First check the purity by measuring the UV at pH 7.0 (use phosphate buffer), and it should have max at 290nm and min at 245nm with a ratio of A290/A250 of 3.7. This ratio goes down to 1.3 as oxidation to the dihydro derivative occurs. The latter can be reduced back to the tetrahydro compound by reaction with 2-mercaptoethanol at room temperature. If oxidation has occurred, then the compound should be chromatographed on DEAE-cellulose (~0.9 milliequiv/g, in AcOform) in (NH4)2CO3 (1.5 M) and washed with 1M NH4OAc containing 0.01M mercaptoethanol till free from UV absorption and then washed with 0.01M mercaptoethanol. All is done in a nitrogen atmosphere. The reduced folate is then eluted with a gradient between 0.01M mercaptoethanol and 1M NH4OAc containing 0.01M mercaptoethanol, and the fractions with absorption at 290nm are collected. These are evaporated under reduced pressure at 25o, and traces of NH4OAc and H2O are removed at high vacuum/25o (~24-48hours). The residue is dissolved in the minimum volume of 0.01M mercaptoethanol, and an equivalent of NaOH is added to convert the acid to the diNa salt and evaporated to dryness at 25o/high vacuum. The pure product has max 290nm ( 32,000) in pH 7.0 buffer. [Sakami Biochemical Preparations 10 103 1963.]