摘要
报道了采用反相高效液相色谱法(RP-HPLC)测定节杆菌(Arthrobacter sp.)黄嘌呤氧化酶活性的有效方法。将酶初提液与含有黄嘌呤的反应体系在37℃下反应20 min,反应终止后通过HPLC测定产物尿酸生成量的变化来分析酶的活性。通过流动相组成、pH和柱温等分离条件的优化,确定了最佳的色谱检测条件以NH4H2PO4(50 mmol/L,pH 7.5)溶液为流动相,流速1 mL/min,柱温25℃,检测波长290 nm。为深入研究微生物细胞内黄嘌呤氧化酶提供了高效检测手段。
A reversed-phase high performance liquid chromatographic method was developed to quanfitate the activity s of xanthine oxidase （XOD, EC 1. 2. 3. 2） involved in nucleic acid metabolism in microbes. Crude enzyme XOD was extracted from the cells of Arthrobacter sp. under ultrasonic incubation, and it catalyzed the oxidation of xanthine into uric acid at 37 ℃ for 20 min. The increment of uric acid in the reaction system was used to calculate the total activity of XOD and assayed by RP-HPLC using a VARIAN MICROSORB-MV 100 - 5 C18 column （250 mm× 4.6 mm i.d., 5 μm）. The optimized HPLC conditions were as follows： mobile phase, 100% NH4H2PO4 （50 mmol/L, pH 7.5）; flow rate, 1 mL/min; column temperature, 25 ℃ and ultraviolet detection wavelength, 290 nm. The correlation coefficient for uric acid within 0.05 - 1.66 mmol/L reached 0.9992. The relative standard deviation for retention time and peak area were 0.10% and 0.12% respectively. The average recovery of xanthine oxidase was 95.75%. The detection limit was 0.01 mmol/L for uric acid. These results demonstrate that this method is simple, rapid and efficient. And it is an alternative potential method for the determination of the activity of XOD in nucleic acid metabolism in microbes.
出处
《分析试验室》
CAS
CSCD
北大核心
2007年第8期41-44,共4页
Chinese Journal of Analysis Laboratory
基金
工业微生物教育部重点实验室基金(KLIB-KF200502)
福建省自然科学基金(C0410006)项目资助
关键词
反相高效液相色谱法
黄嘌呤氧化酶
活力测定
Reversed-phase high performance liquid chromatography
Xanthine oxidase
Activity assay
