亲和色谱和反相高效液相色谱测定菠菜和拟南芥中四氢叶酸代谢物及叶酸的含量(英文)

植物中来源于甘氨酸和丝氨酸的一碳单位转移给四氢叶酸用于四氢叶酸代谢物的生物合成.由于含量低、成份复杂以及稳定性差,植物组织中四氢叶酸代谢物和叶酸的定量分析一度是一个挑战性很强的课题.本研究旨在建立一种可靠方法测定对甲基基团要求不同的植物(例如累积甘氨酸甜菜碱的菠菜与不累积甘氨酸甜菜碱的拟南芥)中四氢叶酸代谢物和叶酸的含量,用于研究这些植物中通过叶酸途径的一碳单位通量.菠菜和拟南芥叶片在金色荧光灯下加液氮研磨,加入大鼠血浆轭合酶粗提物处理,提取物经叶酸结合蛋白琼脂糖亲和色谱柱纯化,用附有荧光和紫外检测器的高效液相色谱仪分离并测定四氢叶酸代谢物和叶酸的含量.菠菜和拟南芥叶片中单谷氨酸型N5-甲基四氢叶酸含量分别是252ng/g和64ng/g,而总N5-甲基四氢叶酸的含量分别是370ng/g和199ng/g.两种植物均检测到少量的四氢叶酸和N5-醛基四氢叶酸,但只在拟南芥叶片而非菠菜叶片中检测到叶酸.实验结果显示,菠菜中单谷氨酸型和多谷氨酸型N5-甲基四氢叶酸的含量均比拟南芥显著增多.这种样品制备和高效液相色谱方法适于测定植物中四氢叶酸代谢物和叶酸的含量.

Analysis of Tetrahydrofolate Metabolites and Folic acid in Spinach and Arabidopsis by Affinity Chromatography and Reversed -Phase High Performance Liquid Chromatography

One-carbon units derived from glycine and serine are transferred to tetrahydrofolic acid (THFA) for biosynthesis of THFA metabolites in plants. Quantitatively analysis of THFA metabolites in plant tissues is challenging because of their low level, complexity and general instability. Our study was to develop a reliable method for quantifying THFA metabolites and folic acid in plants which differ in demand for methyl groups, for example, spinach (an accumulator of glycine betaine) and Arabidopsis thaliana (a non-accumulator of glycine betaine), in preparation for investigation of one-carbon flux through folate pathway in those plants. Under gold fluorescent light, fresh spinach and Arabidopsis leaves were ground in liquid nitrogen and treated with crude preparation of rat plasma conjugase. The resulting extracts were applied to folate binding protein agarose columns for purification. After eluting from the affinity chromatography columns, THFA metabolites and folic acid were determined by reversed-phase HPLC with fluorescence and UV detection. In spinach and Arabidopsis leaves, the contents of mono-glutamate of N5-methyl-tetrahydrofolate (N5-CH3-THFA) were 252 ng/g and 64 ng/g, respectively, while the contents of total N5-CH3-THFA were 370 ng/g and 199 ng/g, respectively. Small amounts of THFA, N5-formyl-tetrahydrofolate (N5-HCO-THFA) were also detected in samples of both plants, folic acid was detected in Arabidopsis leaves, but not in spinach leaves. These results indicate that spinach synthesizes significantly more mono-glutamate and poly-glutamate of N5-CH3-THFA than Arabidopsis does. This method for sample preparation and HPLC analysis is suitable for quantifying THFA metabolites and folic acid in plants.