A rapid radioassay for folic acid reductase and amethopterin

✍ Scribed by Sheldon P. Rothenberg

• Publisher: Elsevier Science
• Year: 1966
• Tongue: English
• Weight: 245 KB
• Volume: 16
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(66)90095-9

✦ Synopsis

The enzymic reduction of isotopically labeled folic acid to tetrahydrofolic acid (H,-folate) employing folic acid reductase extracted from chicken liver has been previously reported from this laboratory (1, 2). Using high specific activity tritiated folic acid (5 to 25 c/mmole) the reduction of substrate levels approximating l&l2 mole was measured. Enzyme activity was assayed in these procedures by measuring the disappearance of the tritiated folic acid substrate, following its separation from the tetrahydrofolate product by paper chromatography. The radioactivity, representing the radiofolate, was assayed directly on the paper strip using a 4-pi gas-flow chromatogram scanner. Although the procedure proved to be precise, several hours was required for completing the chromatography and radioscanning. The purpose of this communication is to report a simple and rapid method of separating the tritiated folate substrate from the tritiated H,-folate product which eliminates paper chromatography and permits use of the more efficient liquid scintillation counting. The reacting mixture contains tritiated folic acid, TPNH, 2-mercaptoethanol, and the enzyme in citrate buffer, pH 4.8, as listed in Table 1. The reaction is stopped at the desired time interval by the addition, in sequence, of stable folic acid and zinc sulfate, the final concentration of each in the reaction mixture being 0.0038 and 0.025 M, respectively. Although these molarities may be varied, the molar ratio