A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase.
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 (K(m) of 0.1 to 10.4 miocroM) 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 miocroM and 0.38+/-0.02 miocromol min(-1)miocrog(-1), respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9+/-9.3 miocroM EM, 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.
Tan
Ying
Y
State Key Laboratory of Element-Organic Chemistry and Department of Chemical Biology, Nankai University,Tianjin 300071, China.
Sun
Lu
L
Xi
Zhen
Z
Yang
Guang-Fu
GF
Jiang
Dong-Qing
DQ
Yan
Xiu-Ping
XP
Yang
Xing
X
Li
He-Yang
HY
D001412:Bacillus subtilis / Q000201:enzymology*; D055162:Biocatalysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D010084:Oxidation-Reduction; D050768:Protoporphyrinogen Oxidase / Q000032:analysis* / Q000378:metabolism; D011524:Protoporphyrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
D011524:Protoporphyrins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C011539:protoporphyrinogen; D050768:Protoporphyrinogen Oxidase