天冬酰胺合成酶B抑制剂高效液相色谱筛选方法的建立与应用

Development and application of screening method for asparagine synthetase B inhibitors by high performance liquid chromatography

建立了一种快速、高效测定天冬酰胺合成酶B(AS-B)酶活性的反相高效液相色谱法(RP-HPLC)。酶反应体系中的氨基酸经2,4-二硝基氟苯(DNFB)柱前衍生,通过RP-HPLC测定酶反应体系前后底物及产物的变化来分析酶的活性。采用的色谱柱为AgilentC18柱(250mm×4.6mm,5μm),以50mmol/L醋酸钠缓冲液(pH6.2)-乙腈(体积比为15∶85)为流动相,流速为1.0mL/min,柱温为30℃,检测波长365nm,于6min内实现了各组分的基线分离。通过该方法测定反应动力学参数进行AS-B的抑制定量分析。将已知AS-B抑制剂L-谷氨酸-γ-甲酯作用于酶反应体系,测得的抑制剂的抑制常数与文献值相接近,证明该方法可用于AS-B抑制剂的筛选。

A screening method for asparagine synthetase B （AS-B） inhibitors by reversedphase high performance liquid chromatography （RP-HPLC） has been established. The contents of asparagines produced in the reaction system can be analyzed by HPLC after the derivatization with 1-fluoro-2,4-dinitrobenzene （DNFB） and used to calculate the total activity of AS-B. The sample was separated on an Agilent C18 column （250 mm ×4.6 mm, 5 μm） at the temperature of 30 ℃ with the elution of 50 mmol/L sodium acetate buffer （ pH 6.2 ）-acetonitrile （ 15 ： 85, v/v） as mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was set at 365 nm. The enzyme reaction system consisted of 100 mmol/L Tris （tris（hydroxymethyl）aminomehane）-HCl buffer （pH 8.0）, 100 mmol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L adenosine triphosphate （ATP）, 10 mmol/L L-aspartate, 10 mmol/L L-glutamine and 2 μg recombinant soybean AS-B （ 1 mL of the total volume）, then mixed for 1 min and incubated for 15 rain at 37 ℃. After quenching with ethanol and centrifugation, the supernatant was derivatized by DNFB and then separated by HPLC. The amino acids in the reaction system were baseline separated within 6 rain. The quantitative analysis of AS-B inhibition was performed by determining its dynamic parameters. The inhibitor L-glutamic acid y-methyl ester was used in the enzyme reaction system to test this method and its inhibition constant obtained was close to the literature value. The established method is fast, accurate, sensitive and suitable for high throughput screening AS-B inhibitors.