【摘要】 报道了一种用反相高效液相色谱法(HPLC)测定丝氨酸:乙醛酸转氨酶(SGAT,EC2.6.1.45)活性变化的有效方法。将酶初提液与含有L 丝氨酸和乙醛酸的反应体系在30℃下反应1h,反应终止后进行2,4 二硝基氟苯衍生化,通过HPLC测定酶反应前后甘氨酸的衍生化产物量的变化来分析酶的活性。通过流动相组成和浓度梯度的优化,既节省了流动相的用量,也避免了气泡的产生。研究结果表明,该测定方法的准确度和灵敏度比分光光度法和纸色谱分析法高,且简便易行;对于其他转氨酶活性的测定也有一定的参考价值。更多还原

【Abstract】 Serine:glyoxylate aminotransferase (SGAT, EC2.6.1.45) is one of the key enzymes involved in the photorespiration pathway of plants. A method for the determination of SGAT activity by reversedphase high performance liquid chromatography (HPLC) with 1fluoro2,4dinitrobenzene (FDNB) precolumn derivatization is reported. The method has simple procedures and is accurate, rapid and efficient. The SGAT catalyzes the transfer of amino group from serine to glyxolate, producing glycine. Therefore, the increase in glycine contents in the reaction system can be analyzed by HPLC after derivatization and used to calculate the total activity of SGAT in the enzyme preparation. SGAT was extracted from plant tissues and an aliquot of the preparation was taken to incubate with Lserine, glyoxylate and pyridoxal 5′phosphate at 30 ℃ for 60 min. The derivatization of the amino acids with FDNB was conducted at 60 ℃ for 60 min. After derivatization the sample was analyzed by HPLC with a C18 column. This method was applied to measure the SGAT activity in duckweed plants and reliable results were obtained. 更多还原