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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 the determination of purine nucleoside phosphorylase (PNP) in the body of Paecilomyces foetida. Inosine was used as a substrate to evaluate its activity. Methods The HPLC conditions were as follows: Ecosil C18-AQ column (250 mm × 4.6 mm, 5 μm), mobile phase consisted of 10 mmol·L-1 ammonium dihydrogen phosphate buffer (p H4.5) , The flow rate was 0.8 m L · min-1, the column temperature was at room temperature and the detection wavelength was 254 nm. The substrate was incubated with crude protein extract for 30 min at room temperature. After boiling for 5 min, the reaction was stopped at 12000 r · min- 1 centrifuged 10 min, the supernatant filtered after HPLC analysis to Origin Pro 8.5 software mapping, enzyme kinetic parameters. Results The enzymatic reaction of hypoxanthine showed a good linear relationship (r = 0.9996) in the range of 0.5-40 μmol·L-1. The precision was less than 3.5% and the recovery was more than 80%. PNP enzyme kinetic parameters: Vmax 0.015nmol · min-1 · mg-1, Km 19.8μmol·L-1. Conclusion The experimental system related to the determination of hypoxanthine metabolism enzyme activity was successfully constructed. The data obtained from this method are stable and reliable and can accurately reflect the dynamic changes of key enzyme activities.