采用ODS分离柱(250 mm×4.6 mm i.d.)、恒组成流动相(8.5% CH3CN-33 mmol·L-1磷酸缓冲液,pH 3.0)并结合荧光检测的高效液相色谱(HPLC)技术,可以对生物体内重要的叶酸存在形态,四氢叶酸和5-甲基四氢叶酸进行分析定量;将这种检测方法应用于亚甲基四氢叶酸还原酶和甲硫氨酸合成酶的活性分析,不需要使用放射性同位素,并且具有检测灵敏度高、特异性强、重复性好、操作简单迅速的特点.使用抗坏血酸、巯基乙醇或二硫苏糖醇,在检测过程中叶酸化合物和酶保持稳定;反应体系中也没有任何试剂会干扰反应产物四氢叶酸和5-甲基四氢叶酸的HPLC检测.
For the determination of tetrahydrofolate (H_4folate) and 5-methyltetrahydrofolate (5-CH_3-H_4folate), a high-performance liquid chromatography (HPLC) system composed of a single reversed-phase octadecylsilane column and fluorescence detector by an isocratic solvent was proposed. This HPLC system perfectly analyzed H_4folate and 5-CH_3-H_4folate in biological samples. We applied this HPLC system into assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring their products of 5-CH_3-H_4folate and H_4folate, to give a new method for determining the enzyme activity without use of radioactive compounds, and the sensitivity of the method is sufficient for measurement of these enzyme activities in crude extracts. Using sodium ascorbate and 2-mercaptoethanol or dithiothreitol,folate derivatives and enzymes were stable in the assay process. No reagents in the assay mixture were found to disturb the separation and detection of both 5-CH_3-H_4folate and H_4folate in our assay system. In addition, our analytical procedure is very simple and economical.