This chapter discusses the enzymic preparations of radiolabeled +-L-5-methyltetrahydrofolate and +-L-formyltetrahydrofolate. The more stable folate coenzymes, 5-formyl-H4folate and 5-methyl-H4folate, may each be synthesized radiolabeled in: (a) a stable portion of the folate molecule and (b) the formyl or methyl adduct. Because the reduction of folic acid to tetrahydrofolic acid introduces asymmetry at carbon-6 and only one of the two possible stereoisomers at this center is enzymically active, the preparations discussed in this chapter use dihydrofolate reductase to catalyze this reaction. The resultant preparations are entirely physiologically active. By mixing the preparations of the 14C- and all-labeled coenzymes, it is possible to follow the fate of both the one-carbon adduct and the folate coenzyme in a single biological experiment. The procedures used in the study result in preparations that have the same specific radioactivities as the incorporated radiolabeled starting materials. In the laboratory, the recovery of radiolabel into final products, in various preparations, has been in the range of 28%-50% for 5-methyl[3H]H4folate, 25%-41% for 5-[14C]methyl-H4folate, 25%-26% for 5-formyl[3H]H4folate, and 14%-15% for 5-[14C]formyl-H4folate. By paper chromatography, preparations of radiolabeled 5-formyl-H4folate have appeared radiochemically pure. Rechromatography on diethylaminoethyl cellulose DEAE-sephadex has allowed more exact estimates of the radiochemical purity of these preparations: 95% for 5-formyl[3H]H4folate, 97% for 5-[14C]formyl-H4folate, 96%-99% for 5-methyl[3H]H4folate, and 95% for 5-[14C]methyl-H4folate.
All Science Journal Classification (ASJC) codes
- Molecular Biology