[195] Dihydrofolic reductase (5,6,7,8-tetrahydrofolate: NADP oxidoreductase, EC 1.5.1.3)

Publisher Summary Dihydrofolic reductase catalyzes the reversible reduction of dihydrofolate by TPNH (triphosphopyridine nucleotide). This chapter focuses on the assay method of dihydrofolic reductase. At neutral pH, the reaction normally proceeds in the forward direction, and the enzymatic activity is assayed by measuring the decrease in absorbancy at 340 nm, which results from both the reduction of dihydrofolate and the oxidation of TPNH. Considerable difficulties are encountered in attempts to assay the reverse reaction colorimetrically, using techniques employed in assaying TPN + - or DPN + -dependent dehydrogenase enzymes. Stabilization of the tetrahydrofolate solutions with antioxidants often results in nonenzymatic reactions with the chromogenic substrates. The forward reaction can be successfully assayed colorimetrically in the presence of the tetrazolium salt 3-(4, 5-dimethylthiazolyl-2)- 2, 5-diphenyl tetrazolium bromide (MTT). The tetrahydrofolate produced by the enzymatic activity reduces the MTT to a formazan having a λ max at 560 nm. The tetrahydrofolate is oxidized to reform dihydrofolate. The method is used to locate dihydrofolate reductase activity following electrophoresis on polyacrylamide gels and cellulose acetate membranes.