A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase

Ying Tan, Lu Sun, Zhen Xi, Guang Fu Yang, Dong Qing Jiang, Xiu Ping Yan, Xing Yang, He Yang Li

Research output: Contribution to journalArticle

Abstract

Protoporphyrinogen oxidase (PPO) is a flavin adenine dinucleotide (FAD)-containing enzyme in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls, which has been identified as one of the most important action targets of commercial herbicides. The literature reports gave different PPO-catalytic kinetic parameters for the substrate protoporphyrinogen IX (Km of 0.1 to 10.4 μM) with different sources of PPO using fluorescent or HPLC methods. Herein we assayed the enzymatic activity of recombinant Bacillus subtilis PPO by using capillary electrophoresis (CE), a method with high separation efficiency, easy automation, and low sample consumption. The Michaelis constant and maximum reaction velocity were determined as 7.0 ± 0.6 μM and 0.38 ± 0.02 μmol min-1 μg-1, respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9 ± 9.3 μM{cyrillic}. The relationship between cofactor FAD and PPO activity can also be quantitatively studied by this CE method. The CE method used here should also be a convenient, reliable method for PPO study.

Original languageEnglish (US)
Pages (from-to)200-204
Number of pages5
JournalAnalytical Biochemistry
Volume383
Issue number2
DOIs
StatePublished - Dec 15 2008
Externally publishedYes

Fingerprint

Protoporphyrinogen Oxidase
Capillary electrophoresis
Capillary Electrophoresis
Bacilli
Bacillus subtilis
Assays
Flavin-Adenine Dinucleotide
Herbicides
Tetrapyrroles
Automation
Biosynthetic Pathways
Chlorophyll
Heme
Kinetic parameters
High Pressure Liquid Chromatography

Keywords

  • Capillary electrophoresis
  • FAD
  • Herbicide
  • Kinetics
  • Protoporphyrinogen oxidase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tan, Y., Sun, L., Xi, Z., Yang, G. F., Jiang, D. Q., Yan, X. P., ... Li, H. Y. (2008). A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase. Analytical Biochemistry, 383(2), 200-204. https://doi.org/10.1016/j.ab.2008.09.023

A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase. / Tan, Ying; Sun, Lu; Xi, Zhen; Yang, Guang Fu; Jiang, Dong Qing; Yan, Xiu Ping; Yang, Xing; Li, He Yang.

In: Analytical Biochemistry, Vol. 383, No. 2, 15.12.2008, p. 200-204.

Research output: Contribution to journalArticle

Tan, Y, Sun, L, Xi, Z, Yang, GF, Jiang, DQ, Yan, XP, Yang, X & Li, HY 2008, 'A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase', Analytical Biochemistry, vol. 383, no. 2, pp. 200-204. https://doi.org/10.1016/j.ab.2008.09.023
Tan, Ying ; Sun, Lu ; Xi, Zhen ; Yang, Guang Fu ; Jiang, Dong Qing ; Yan, Xiu Ping ; Yang, Xing ; Li, He Yang. / A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase. In: Analytical Biochemistry. 2008 ; Vol. 383, No. 2. pp. 200-204.
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