Direct spectrophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate.

A direct spectrophotometric method has been devised to follow the oxidation of tetrahydrofolate during the synthesis of thymidylate. It is based on the marked spectral change which occurs when 5, IO-methylenetetrahydrofolate is converted to dihydrofolate. A nonenzymatic reaction between formaldehyde and tetrahydrofolate yields 5,lO -methylenetetrahydrofolate (also called hydroxymethyltetrahydrofolate) (8, 9). The instability of tetrahydrofolate is well known; however, in the presence of an excess of formaldehyde and a high concentration of 2-mercaptoethanol (0.1 M) there is essentially no degradation of 5, lo-methylenetetrahydrofolate for a period of 1 hour at room temperature. The spectra of tetrahydrofolate and dihydrofolate in the presence of formaldehyde and 2-mercaptoethanol are shown in Fig. 1. Of special note is the difference spectrum, Fig. 1 (Curve C), characterized by an increase in absorbancy at 338 mp (Ae = SSOO), and a decrease in absorbancy at 303 rnp ( -Ae = 9000). An identical difference spectrum was obtained with a reaction mixture consisting of deoxyuridylate, tetrahydrofolate, formaldehyde, Mg++, 2-mercaptoethanol, and purified Escherichiu coli thymidylate synthetase (Fig. 2). The spectral change which occurred during the enzymatic oxidation of 5, lo-methylenetetrahydrofolate could be correlated with the synthesis of P32-labeled thymidylate (Table I). For each mole of thymidylate formed, 1 mole of dihydrofolate appeared as measured by the increase in absorbancy at 340 rnp. The product of the enzymatic oxidation of tetrahydrofolate was adsorbed on DEAE-cellulose and recovered in excellent yield by gradient elution with Tris (13). Deoxyuridylate (0.94 pmole), dl, n-tetrahydrofolate (2.2 pmoles) and E. coli thymidylate synthetase (2.2 mg) in 11 ml of Mixture I (Fig. 1) were incubated for 60 minutes at room temperature. The increase in absorbancy at 340 rnp was 0.540, equivalent to the formation of 0.93 pmole of dihydrofolate. The reaction mixture was then poured through a DEAE-cellulose column (1.8 x 10 cm) and