4
M. Pohanka & O. Holas
J Enzyme Inhib Med Chem, Early Online: 1–4
14. Collier HB. Letter: a note on the molar absorptivity of reduced
Anal
Conclusions
Ellman’s
Biochem 1973;56:310–11.
reagent,
3-carboxylato-4-nitrothiophenolate.
In this article, 2,6-dichlorophenolindophenol acetate was per-
formed as an chromogenic substrate for AChE and compared to
the standard Ellman’s method. The finding presented here
encourage authors to claim that 2,6-dichlorophenolindophenol
acetate is suitable for routine assay where AChE activity should
be performed. Assay based on 2,6-dichlorophenolindophenol
acetate is not sensitive to interferences under the conditions
tested. Moreover, the assay uses long wavelength for absorbance
measurement, hence typical interferents such as hemoglobin do
not overlap with the assay comparing to the Ellman’s method.
Lower turnover rate when compared to acetylthiocholine is major
disadvantage of the method and it is necessary to plan long time
intervals for coloration arising.
15. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman’s
reagent. Meth Enzymol 1983;91:49–60.
16. Bissbort SH, Vermaak WJH, Elias J, et al. Novel test and its
automation for the determination of erythrocyte acetylcholinesterase
and its application to organophosphate exposure. Clin Chim Acta
2001;303:139–45.
17. Trott O, Olson AJ. AutoDock Vina: improving the speed and
accuracy of docking with
a new scoring function, efficient
optimization, and multithreading. J Comput Chem 2010;31:455–61.
18. Schuttelkopf AW, van Aalten DM. PRODRG: a tool for high-
throughput crystallography of protein-ligand complexes. Acta
Cristallographica D Biological Crystallogra 2004;60:1355–63.
19. Radic Z, Pickering NA, Vellom DC, et al. Three distinct domains in
the cholinesterase molecule confer selectivity for acetyl- and
butyrylcholinesterase inhibitors. Biochemistry 1993;32:12074–84.
20. Nair HK, Seravalli J, Arbuckle T, Quinn DM. Molecular recognition
in acetylcholinesterase catalysis: free-energy correlations for sub-
strate turnover and inhibition by trifluoro ketone transition-state
analogs. Biochemistry 1994;33:8566–76.
Acknowledgements
The European Union is gratefully acknowledged for project TEAB;
CZ.1.07/2.3.00/20.0235. A long-term organization development plan
1011 (Faculty of Military Health Sciences, University of Defence, Czech
Republic) is acknowledged as well.
21. Masson P, Schopfer LM, Bartels CF, et al. Substrate activation in
acetylcholinesterase induced by low pH or mutation in the pi-cation
subsite. Biochim Biophys Acta-Protein Struct Molec Enzym 2002;
1594:313–24.
Declaration of interest
22. Masson P, Nachon F, Bartels CF, et al. High activity of human
butyrylcholinesterase at low pH in the presence of excess
butyrylthiocholine. Eur J Biochem 2003;270:315–24.
23. Visciarelli EC, Chopa CS, Picollo MI, Ferrero AA. Cholinesterase
activity during embryonic development in the blood-feeding bug
Triatoma patagonica. Med Vet Entomol 2011;25:297–301.
24. Ciliv G, Ozand PT. Human erythrocyte acetylcholinesterase puri-
fication, properties and kinetic behavior. Biochim Biophys Acta
1972;284:136–56.
The authors declare no conflicts of interests. The authors alone
are responsible for the content and writing of this article.
References
1. Pohanka M. Acetylcholinesterase inhibitors: a patent review (2008 -
present). Expert Opin Ther Pat 2012;22:871–86.
25. Walsh SB, Dolden TA, Moores GD, et al. Identification and
characterization of mutations in housefly (Musca domestica)
acetylcholinesterase involved in insecticide resistance. Biochem J
2001;359:175–81.
2. Pohanka M. Cholinesterases, a target of pharmacology and toxicol-
ogy. Biomed Pap 2011;155:219–29.
3. de los Rios C. Cholinesterase inhibitors: a patent review (2007–
2011). Expert Opin Ther Pat 2012;22:853–69.
26. Shi J, Boyd AE, Radic Z, Taylor P. Reversibly bound and covalently
attached ligands induce conformational changes in the omega loop,
Cys69-Cys96, of mouse acetylcholinesterase. J Biol Chem 2001;
276:42196–204.
4. Li B, Duysen EG, Carlson M, Lockridge O. The butyrylcholinester-
ase knockout mouse as a model for human butyrylcholinesterase
deficiency. J Pharmacol Exp Ther 2008;324:1146–54.
5. Pohanka M. Butyrylcholinesterase as a biochemical marker, a
review. Brat Med J 2013;114:726–34.
6. Pohanka M. Spectrophotomeric assay of aflatoxin B1 using
acetylcholinesterase immobilized on standard microplates. Anal
Lett 2013;46:1306–15.
7. Pohanka M. Acetylcholinesterase based dipsticks with indoxylace-
tate as a substrate for assay of organophosphates and carbamates.
Anal Lett 2012;45:367–74.
8. Ellman GL, Courtney KD, Andres Jr. V, Feather-Stone RM. A new
and rapid colorimetric determination of acetylcholinesterase activity.
Biochem Pharmacol 1961;7:88–95.
9. Arduini F, Errico I, Amine A, et al. Enzymatic spectrophotometric
method for aflatoxin B detection based on acetylcholinesterase
inhibition. Anal Chem 2007;79:3409–15.
10. Vasudevan G, McDonald MJ. Wavelength-dependent spectral
changes accompany CN-hemin binding to human apohemoglobin.
J Prot Chem 2000;19:583–90.
11. Pohanka M. Cholinesterases in biorecognition and biosensor con-
struction, a review. Anal Lett 2013;46:1849–68.
12. Sinko G, Calic M, Bosak A, Kovarik Z. Limitation of the Ellman
method: cholinesterase activity measurement in the presence of
oximes. Anal Biochem 2007;370:223–7.
27. Miao Y, He N, Zhu JJ. History and new developments of assay for
cholinesterase activity and inhibition. Chem Rev 2010;110:5216–34.
28. Michelet F, Gueguen R, Leroy P, et al. Blood and plasma glutathione
measured in healthy subjects by HPLC: relation to sex, aging,
biological variables, and life habits. Clin Chem 1995;41:1509–17.
29. Taravati A, Ardestani SK, Soroush MR, et al. Serum albumin and
paraoxonase activity in Iranian veterans 20 years after sulfur mustard
exposure. Immunopharmacol Immunotoxicol 2012;34:706–13.
30. Bhonsle HS, Korwar AM, Kote SS, et al. Low plasma albumin levels
are associated with increased plasma protein glycation and HbA1c in
diabetes. J Proteome Res 2012;11:1391–6.
31. Vanderjagt DJ, Garry PJ, Hunt WC. Ascorbate in plasma as
measured by liquid-chromatography and by dichlorophenolindophe-
nol colorimetry. Clin Chem 1986;32:1004–6.
32. Chung WY, Chung JKO, Szeto YT, et al. Plasma ascorbic acid:
measurement, stability and clinical utility revisited. Clin Biochem
2001;34:623–7.
33. Merola ET, Catherman AD, Yehl JB, Strein TG. Determination of
total antioxidant capacity of commercial beverage samples by
capillary electrophoresis via in-line reaction with 2,6 – dichlor-
ophenolindophenol. J Agric Food Chem 2009;57:6518–23.
13. McGarry KG, Bartlett RA, Machesky NJ, et al. Evaluation of 34. Azarkina N, Konstantinov AA. Stimulation of menaquinone-
dependent electron transfer in the respiratory chain of Bacillus
subtilis by membrane energization. J Bacteriol 2002;184:5339–47.
HemogloBindÔ treatment for preparation of samples for cholin-
esterase analysis. Adv Biosci Biotechnol 2013;4:1020–3.