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 journalArticlepeer-review

22 Scopus citations


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
Issue number2
StatePublished - Dec 15 2008
Externally publishedYes


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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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