反相高效液相色谱法测定磷酸果糖激酶的活性

Determination of Phosphofructokinase Activity by Reversed-Phase High Performance Liquid Chromatography

采用反相高效液相法（RP-HPLC）测定磷酸果糖激酶的活性。将磷酸果糖激酶与含有果糖-6-磷酸和ATP的反应体系混合，置于30℃水浴中，反应20min后，沸水浴3min终止反应。通过RP-HPLC测定酶反应前后产物二磷酸腺苷（ADP）量的变化来分析酶的活性。ADP的生成量在5～400mg／L范围内具有良好的线性关系，方法的精密度良好（RSD〈5％）。本方法与通常所用的酶偶联法（ECM）相比准确度高，且简便易行。

Phosphofructokinase （PFK, EC2. 7. 1. 11） activity was determined by reversed-phase high performance liquid chromatography（RP-PHLC） in this paper. After PFK, Fructo-6-phospho and ATP were mixed in the reaction system, the mixture was incubated at 30℃ for 30 min, and the reaction was stopped in the boiling water for 3 rain. The quantity of product ADP before and after reaction which was determined by RP-PHLC was dependent on the PFK activity. The product ADP had good linear relationship at 5～400 mg/L with a good precision （RSD〈5%）. RP-PHLC was more accurat than the conventional enzyme coupling method （ECM）, and its operation was more convenient.