HPLC separation of the reaction mixture in mammalian cell catalyzed by themultienzyme

The product of the first three steps of de novo pathway of pyremidine biosy nthesis in mammalian cell catalyzed by carbamoyl phosphate synthetase - asp arate transcarbamoylase - dihyroorotase are adenosine diphosphate(ADP) and dihydroorotate(DHO). And one of reactant is adenosine triphosphate(Am). A I -FPLC method for separation of ADP, ATP and DHO was developed. A Zarbox col umn (150 mm x 4.6 mm I.D,) was used. The mobile phase were 0.01 mol.L-1 pot assium dihydrogen phosphate buffer( pH 4.7) and 0.5 mol.L-1 potassium dihyd rogen phosphate(pH 4.6). The gradient was programmed at 0 similar to 5 min, (psi = 1, volume ratio) 0.01 mol.L-1 KH2PO4 then 5 similar to 50 min, (psi = 1, volume ratio) 0.01 mol.L-1 KH2PO4 to (psi = 1, volume ratio) 0.5 mol. L-1 KH2PO4. The flow rate was 0,7 mL.min(-1). The optimum detection wavelen gth and the sequence of ATP, ADP and DHO peaks were detected. The working c ursor was plotted and then the corresponding concentrations of ATP, ADP and DHO in the catalytic reaction were calculated.