COFACTOR ROLE FOR 10-FORMYLDIHYDROFOLIC ACID

10-Formyl-7,8-dihydrofolic acid (10-HCO-H(2)folate) was prepared by co ntrolled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-H CO-H(4)folate). The UV spectra of the 10-HCO-H(2)folate preparation ha s lambda(max.) 234, 333 nm and lambda(min.) 301 nm at pH 7.4, and lamb da(max.) 257, 328 nm and lambda(min.) 229, 307 nm at pH 1. H-1-NMR spe ctroscopy of 10-HCO-H(2)folate (in (H2O)-H-2; 300 MHz) suggested a pur e compound and gave resonances for one formyl group proton, two proton s on C-7 and C-9, and no evidence for a C-6 proton, which is consisten t with the structure proposed. The spectral properties indicated that the 10-HCO-H,folate preparation is not appreciably contaminated with 1 0-HCO-H(4)folate, 5,10-methenyltetrahydrofolic acid (5,10-CH=H(4)folat e) or 10-formylfolic acid (10-HCO-folate). The above data establish th at the 10-HCO-H(2)folate prepared here is authentic. In contrast, a fo late with a UV spectrum having lambda(max.) 272 nm and lambda(min.) 25 6 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H(4)folate and reported to be 97% pure [Baram, Chabner, Drak e, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H(2)folate. 10-HCO-H-2 folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazole carboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-aminoimidazole-4-carboxamide ribotide (AICAR ) resulting in the appearance of approximately 1 mol of H(2)folate pro duct for each mol of AICAR formylated. The present 10-HCO-H(2)folate p reparation had a kinetic advantage over 10-HCO-H(4)folate resulting fr om a difference of approx. 5-fold in K-m values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H(2)folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, E C 2.1.2.2). The existence in vivo of 10-HCO-H(2)folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10- HCO-folate in bile and urine after administration of radiolabelled fol ic acid.