参环毛蚓体内核苷磷酸化酶的活性测定及动力学分析

Assay of Purine Nucleoside Phosphorylase Activity in Pheretima Asperfillum(E·Perrier) and Analysis of Its Enzyme Kinetics

目的建立参环毛蚓体内嘌呤核苷磷酸化酶（PNP）的检测方法,以肌苷为底物,评价该酶的活性。方法采用HPLC法,色谱条件：Ecosil C18-AQ柱（250 mm×4.6 mm,5μm）,流动相为10 mmol·L^-1磷酸二氢铵缓冲液（p H4.5）和甲醇,梯度洗脱,流速为0.8 m L·min^-1,柱温为室温,检测波长254 nm,用底物肌苷与组织粗蛋白提取液在室温孵育30 min,沸水煮5 min终止反应,12000 r·min^-1离心10 min,取上清过滤后进行HPLC分析,以Origin Pro 8.5软件作图,计算酶动力学参数。结果酶反应中产物次黄嘌呤在0.5-40μmol·L^-1范围内呈良好的线性关系（r=0.9996）;精密度小于3.5%,回收率大于80%。PNP酶动力学参数：Vmax为0.015nmol·min^-1·mg^-1,Km为19.8μmol·L^-1。结论成功构建了参环毛蚓体内次黄嘌呤代谢酶活性测定相关的实验体系,该法所得数据稳定可靠,可准确反映关键酶活性的动态变化。

Objective To establish a method for evaluating the purine nucleoside phosphorylase（PNP） activity in Phe re tima aspe rfillum（E. Perrier）by high-performance liquid chromatography（HPLC）-UV detection,and to analyse the enzyme kinetics of PNP. Methods Ecosil C18-AQ（250 mm ×4.6 mm, 5 μm） column, a mobile phase of 10mmol·L^-1ammonium dihydrogen phosphate buffer（p H4.5）-methyl alcohol, and gradient elution were used. The flow rate was 0.8 m L·min^-1,and detection wavelength was 254 nm. The samples were incubated with inosine respectively for 30 min at room temperature,and the reactions were terminated by immersion of the test-tubes in boiling water for 5min. The reactive liquid was centrifuged at 12000 r·min-1for 10 min. Finally,the supernatant was analyzed by HPLC,and the kinetic parameters were calculated with Origin Pro 8.5 software. Results The linear range of the reactive product of hypoxanthine was 0.5-40 μmol·L-1（r=0.9996）and the detection limit was 0.05μmol·L^-1. The relative standard deviations were less than 3.5 %,and the method recovery was more than 80 %. The results of enzyme kinetic analysis of PNP showed that that Vmax was 0.015 nmol·min-1·mg-1,and Km was 19.8 μmol·L^-1. Conclusion The experimental system for determing enzyme activity in purine metabolism of Pheretima aspe rfillum has been established successfully. The established method is steady, accurate and suitable for assaying the dynamic changes of PNP activity,which will lay an experimental basis for the further study of purine metabolism and its exact mechanism in the future.