哺乳动物细胞中所得多肽复合酶CAD催化反应产物的HPLC分析

HPLC Separation of the Reaction Mixture in Mammalian Cell Catalyzed by the Multienzyme

本研究报道了以150 mm×4.6 mmI.D.Zorbax为色谱柱,10 mmol@L-1KH2PO4和500mmol@L-1KH2PO4水溶液为洗脱液,梯度程序为0～5 min(φ=1,体积分数)10 mmol@L-1KH2PO4溶液,5～50min由(φ=1)10 mmol@L-1KH2PO4溶液转换到(φ=1)500mmol@L-1KH2PO4溶液时,高效液相色谱分离了哺乳动物细胞中所得多肽复合酶CAD催化反应混合物三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和二氢乳清酸(DHO),确定了ATP,ADP和DHO的出峰顺序,制作了工作曲线,确定了催化反应混合物中ATP,ADP和DHO的相应浓度.

The product of the first three steps of de novo pathway of pyremidine biosynthesis in mammalian cell catalyzed by carbamoyl phosphate synthetase - asparate 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 column (150 mm x 4.6 mm I.D,) was used. The mobile phase were 0.01 mol.L-1 potassium dihydrogen phosphate buffer( pH 4.7) and 0.5 mol.L-1 potassium dihydrogen 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 wavelength and the sequence of ATP, ADP and DHO peaks were detected. The working cursor was plotted and then the corresponding concentrations of ATP, ADP and DHO in the catalytic reaction were calculated.