Journal
TALANTA
Volume 232, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2021.122444
Keywords
Carbonic anhydrase IX; Inhibitor screening; Capillary electrophoresis; Electrophoretically mediated microanalysis; Traditional Chinese medicine
Categories
Funding
- National Natural Science Foundation of China [82073808, 81872828]
Ask authors/readers for more resources
The study developed an EMMA method which combines TDLFP and rapid polarity switching technology to achieve rapid mixing of different reactants. By optimizing the enzyme reaction and separation conditions, the effectiveness of this method for screening carbonic anhydrase IX inhibitors was successfully validated.
An electrophoretically mediated microanalysis (EMMA) method for the screening of carbonic anhydrase IX inhibitors in traditional Chinese medicine (TCM) was developed. This method combines transverse diffusion of laminar flow profiles (TDLFP) and rapid polarity switching technology to achieve rapid mixing of different reactants. Different electromigration rates of different substances make it possible that incubation, separation and detection are carried out continuously in a same fused-silica capillary. In this experiment, p-nitrophenyl acetate (pNPA) was used as the substrate for the enzyme reaction, which solved the problem that capillary electrophoresis could not detect carbonate, carbon dioxide, etc., the conventional substrates of carbonic anhydrase IX. After optimizing the enzyme reaction and separation conditions, the separation of substrate and product can be finished by baseline within 4 min. The Michaelis constant of carbonic anhydrase IX was measured to be 1.2 mM. A known inhibitor acetazolamide was used to evaluate the feasibility of this method for screening carbonic anhydrase IX inhibitors, and the half-maximal inhibitory concentration (IC50) was calculated to be 1.26 mu M. Finally, 4 natural compounds of 15 traditional Chinese medicine standards were discovered to exhibit enzyme inhibitory activity, including polydatin, matrine, dauricine and cepharanthine, proving that the EMMA method is an effective means for screening carbonic anhydrase IX inhibitors. The results were supported by molecular docking study.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available