2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数

引用本文: 胡玥, 王雪娇, 李恒, 高文运. 2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数[J]. 分析化学, 2012, 40(12): 1859-1864. doi: 10.3724/SP.J.1096.2012.20447 shu

Citation: HU Yue, WANG Xue-Jiao, LI Heng, GAO Wen-Yun. Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose- 5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent[J]. Chinese Journal of Analytical Chemistry, 2012, 40(12): 1859-1864. doi: 10.3724/SP.J.1096.2012.20447 shu

2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数

通讯作者: 高文运

基金项目:
本文系国家自然科学基金项目(No.21172179) (No.21172179)

西北大学研究生创新基金(No.10YYB02)资助 (No.10YYB02)

摘要: 利用含羰基化合物与2,4-二硝基苯肼在酸性条件下反应得到的产物腙对紫外-可见光有吸收的特性,采用柱前衍生高效液相色谱法测定了1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数。酶反应产物1-脱氧-D-木酮糖-5-磷酸在碱性磷酸酶的作用下生成去磷酸化产物1-脱氧-D-木酮糖,然后与2,4-二硝基苯肼衍生成腙。衍生化反应的适宜条件为:HClO₄浓度为1.5%,反应温度37℃,反应时间60 min,2,4-二硝基苯肼与1-脱氧-D-木酮糖-5-磷酸的摩尔比为6:1。色谱条件:0-17 min,40%-80%甲醇;18-20 min,40% 甲醇。结果表明,本方法具有较高的灵敏度和良好的线性关系,1-脱氧-D-木酮糖-5-磷酸的检出限为1.0 mg/L,在0.005-1.0 g/L范围内线性相关系数为0.999,相对标准偏差小于5.0%(n=3),标准回收率为102.22%。用本方法测得的1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数K_m、V_max和K_cat与文献报道一致。

关键词:

English

Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose- 5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent

1.
College of Life Sciences, Northwest University, Xi'an 710069, China

Corresponding author: 高文运

Received Date: 27 April 2012
Fund Project:
本文系国家自然科学基金项目(No.21172179)

西北大学研究生创新基金(No.10YYB02)资助

Abstract: Carbonyl containing compounds can normally be determined by high performance liquid chromatography (HPLC) using pre-column derivatization with 2,4-dinitrophenylhydrazine (DNPH). Using this method, the steady-state kinetic parameters of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) were measured. First, the enzymatic product 1-deoxy-D-xylulose-5-phosphate (DXP) was dephosphorylated by alkaline phosphatase, then the product 1-deoxy-D-xylulose (DX) was derived with DNPH in acidic solution to give the corresponding hydrazones which was subsequently determined by HPLC. The optimum derivatization conditions were as follows: acidity 1.5% perchloric acid, reaction temperature 37℃, reaction time of 60 min, molar ratio of DNPH to DXP 6:1. The HPLC was run with a linear gradient of methanol-water solvent system: 0 min, 40% methanol; 17 min, 80% methanol; 18 min, 40% methanol; 20 min, 40% methanol. The method has a detection limit of 1 mg/L for DXP and the linear correlation coefficient in the range of 0.005-1 g/L was 0.999. The relative standard deviation is less than 5.0%. The steady-state kinetic parameters of DXS determined with this method are identical with the reported data.

Key words:

Non-mevalonate pathway
/ 1-Deoxy-D-xylulose-5-phosphate synthase
/ Kinetic parameters
/ 2,4-Dinitrophenylhydrazine
/ High performance liquid chromatography

1. [1]
  1 Eisenreich W. Bacher A, Arigoni D, Rohdich F. Cell. Mol. Life Sci., 2004, 61(5): 1401-14261 Eisenreich W. Bacher A, Arigoni D, Rohdich F. Cell. Mol. Life Sci., 2004, 61(5): 1401-1426
2. [2]
  2 Rohmer M. In Comprehensive Natural Products II, Chemistry and Biology, Mander L, Liu H W. Eds. Elsevier, 2010, 1: 517-5552 Rohmer M. In Comprehensive Natural Products II, Chemistry and Biology, Mander L, Liu H W. Eds. Elsevier, 2010, 1: 517-555
3. [3]
  3 Hunter W. J. Biol. Chem., 2007, 282(30): 21573-215773 Hunter W. J. Biol. Chem., 2007, 282(30): 21573-21577
4. [4]
  4 JIN Rong, ZHU Chang-Qing, XU Chang-Jie. Chinese J. Cell Biol., 2007, 29(9): 706-7124 JIN Rong, ZHU Chang-Qing, XU Chang-Jie. Chinese J. Cell Biol., 2007, 29(9): 706-712
5. [5]
  金蓉, 朱长青, 徐昌杰. 细胞生物学杂志, 2007, 29(9): 706-712金蓉, 朱长青, 徐昌杰. 细胞生物学杂志, 2007, 29(9): 706-712
6. [6]
  5 Fazia B, Naima H, Odile M, Franoise S, Michel H, Paul M V, Philippe L. Eur. J. Pharmacol., 2004, 485(3): 227-2345 Fazia B, Naima H, Odile M, Franoise S, Michel H, Paul M V, Philippe L. Eur. J. Pharmacol., 2004, 485(3): 227-234
7. [7]
  6 Lange B M, Wildung M R, David M, Rodney C. Proc. Natl. Acad. Sci. USA, 1998, 95(5): 2100-21046 Lange B M, Wildung M R, David M, Rodney C. Proc. Natl. Acad. Sci. USA, 1998, 95(5): 2100-2104
8. [8]
  7 Querol J, Besumbes O, Lois L M, Boronat A, Imperial S. Anal. Biochem., 2001, 296(9): 101-1057 Querol J, Besumbes O, Lois L M, Boronat A, Imperial S. Anal. Biochem., 2001, 296(9): 101-105
9. [9]
  8 Chahed K, Oudin A, Guivarc'h N, Guivarc'h N, Hamdi S, Chénieux J C, Rideau M, Clastre M. Plant Physio. Biochem., 2000, 38(3): 559-5668 Chahed K, Oudin A, Guivarc'h N, Guivarc'h N, Hamdi S, Chénieux J C, Rideau M, Clastre M. Plant Physio. Biochem., 2000, 38(3): 559-566
10. [10]
  9 Feurle J, Jomaa H, Wilhelm M, Gutsche B, Herderich M. J. Chromatogr. A, 1998, 803(2): 111-1199 Feurle J, Jomaa H, Wilhelm M, Gutsche B, Herderich M. J. Chromatogr. A, 1998, 803(2): 111-119
11. [11]
  10 Han Y S, Cesare S, Robert H, Verpoorte R. J. Chromatogr. A, 2003, 986(2): 291-29610 Han Y S, Cesare S, Robert H, Verpoorte R. J. Chromatogr. A, 2003, 986(2): 291-296
12. [12]
  11 Altincicek B, Hintz M, Sanderbrand S, Wiesner J. FEMS Microbio. Lett., 2000, 190(2): 329-33311 Altincicek B, Hintz M, Sanderbrand S, Wiesner J. FEMS Microbio. Lett., 2000, 190(2): 329-333
13. [13]
  12 Brammer L A, Caren F M. Org. Lett., 2009, 11(20): 4748-475112 Brammer L A, Caren F M. Org. Lett., 2009, 11(20): 4748-4751
14. [14]
  13 Zhou Y F, Cui Z, Li H, Tian J, Gao W Y. Bioorg. Chem., 2010, 38(3): 120-12313 Zhou Y F, Cui Z, Li H, Tian J, Gao W Y. Bioorg. Chem., 2010, 38(3): 120-123
15. [15]
  14 Li H, Tian J, Wang H, Yang S Q, Gao W Y. Helv. Chim. Acta, 2010, 93(9): 1745-175014 Li H, Tian J, Wang H, Yang S Q, Gao W Y. Helv. Chim. Acta, 2010, 93(9): 1745-1750
16. [16]
  15 Shigehisa U, Yohei I, Naoki K. J. Chromatogr. B, 2011, 879(11): 1282-128915 Shigehisa U, Yohei I, Naoki K. J. Chromatogr. B, 2011, 879(11): 1282-1289
17. [17]
  16 Zhu Ya-Mei, Cui Qun, Wang Hai-Yan. Chinese Journal of Chromatography, 2010, 28(1): 159-16316 Zhu Ya-Mei, Cui Qun, Wang Hai-Yan. Chinese Journal of Chromatography, 2010, 28(1): 159-163
18. [18]
  朱鸭梅, 崔群, 王海燕. 色谱, 2010, 28(1): 159-163朱鸭梅, 崔群, 王海燕. 色谱, 2010, 28(1): 159-163
19. [19]
  17 YAN Kun-Ping, JING Xiao-Dan, HAN Jing, DAN Ning, CHEN Chao. Chinese J. Anal. Chem., 2009, 37(10): 1515-151817 YAN Kun-Ping, JING Xiao-Dan, HAN Jing, DAN Ning, CHEN Chao. Chinese J. Anal. Chem., 2009, 37(10): 1515-1518
20. [20]
  严坤平, 景小丹, 韩静, 但宁, 陈超. 分析化学 , 2009, 37(10): 1515-1518严坤平, 景小丹, 韩静, 但宁, 陈超. 分析化学 , 2009, 37(10): 1515-1518
21. [21]
  18 WANG Jing-Yan, ZHU Sheng-Geng, XU Chang-Fa. Biochemistry. Beijing: Higher Education Press, 2002: 355-36318 WANG Jing-Yan, ZHU Sheng-Geng, XU Chang-Fa. Biochemistry. Beijing: Higher Education Press, 2002: 355-363
22. [22]
  王镜岩, 朱胜庚, 徐长法. 生物化学. 北京: 高等教育出版社, 2002: 355-363王镜岩, 朱胜庚, 徐长法. 生物化学. 北京: 高等教育出版社, 2002: 355-363

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数

通讯作者: 高文运

English

Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose- 5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent

Corresponding author: 高文运

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

2,4-二硝基苯肼柱前衍生高效液相色谱法测定1-脱氧-D-木酮糖-5-磷酸合酶稳态动力学参数

通讯作者: 高文运

English

Determination of Steady-State Kinetic Parameters of 1-Deoxy-D-xylulose- 5-phosphate Synthase by Pre-column Derivatization High Performance Liquid Chromatography Using 2,4-Dinitrophenylhydrazine as Derivative Reagent

Corresponding author: 高文运

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs