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目的:利用高效液相色谱法测定叶酸-青霉素G酰化酶(PGA)对N-苯乙酰化阿霉素(DOXP)的催化活性,为叶酸导向的酶催化前体药物的肿瘤治疗(FDEPT)研究奠定理论基础。方法:色谱柱为Diamonsil C18(250 mm×4.6 mm,5μm);流动相为乙腈-水,用85%磷酸调节pH至2.4;梯度程序:0~3 min为24%乙腈,3~15min为80%乙腈;紫外检测波长:495 nm;流速:1 mL/min。结果:DOXP和酶解产物阿霉素的tR分别为5.5 min和12.3 min,原酶PGA和叶酸-PGA偶联酶对DOXP的Km和vmax分别为15μmol/mL,0.094μmol/(min.mg)和19μmol/mL,0.086μmol/(min.mg)。结论:DOXP为PGA的较好底物,叶酸与PGA偶联对酶的催化活性的影响较小。
OBJECTIVE: To determine the catalytic activity of folic acid-penicillin G acylase (PGA) for N-phenylacetylated doxorubicin (DOXP) by high performance liquid chromatography (HPLC) Research laid the theoretical foundation. Methods: The column was Diamonsil C18 (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile-water and the pH was adjusted to 2.4 with 85% phosphoric acid. The gradient program consisted of 24% acetonitrile at 0-3 min and 80 at 3-15 min % Acetonitrile; UV detection wavelength: 495 nm; flow rate: 1 mL / min. Results: The tR values of DOXP and DOX were 5.5 and 12.3 min, respectively. The Km and vmax values of DOXP and PGA were respectively 15μmol / mL and 0.094μmol / (min.mg) And 19 μmol / mL, 0.086 μmol / (min.mg). Conclusion: DOXP is a good substrate for PGA, and the effect of folic acid and PGA conjugation on the catalytic activity of the enzyme is small.