Colorimetric determination of cholinesterase activity – Ellman's assay
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) efficiently catalyze the
hydrolysis of acetylthiocholine (ATCh) – sulfur analog of the natural substrate of these
enzymes. Upon hydrolysis, this substrate analog produces into acetate and thiocholine.
Thiocholine, in the presence of the highly reactive dithiobisnitro-benzoate (DTNB) ion
generates a yellow color, which is visible and can be quantitatively monitored by
spectrophotometric absorption at 405 nm. In order to differentiate between
acetylcholinesterase and butyrylcholinesterase we will use a specific inhibitor against
BChE (5).
We will perform the Ellman assay in 96-well microtiter plates in a final reaction volume
of 200 μl. Substrate hydrolysis is monitored by repeated spectrophotometometric
measurements at 2 min. intervals by a computer-controlled microtiter plate reader which
automatically computes mOD405/min for the best fit straight line through the data points.
We will then convert out data into the standardized units of nanomoles substrate
hydrolyzed per minute per ml of plasma using the extinction coefficient for the yellow
product (13,600 M-1cm-1). The light path length is 0.5 cm. The equation describing
absorbance is A = ε*c*l (also known as Beer-Lambert law), where:
A – absorbance measured in OD (Optical Density units)
ε – epsilon, the molar extinction coefficient which evaluates is how much light will be
absorbed by 1 cm of a 1 M solution of this chemical. Its value depends on what the
chemical is and also on what wavelength (or color) of light is being used.
c – concentation measured in molarity.
l – light path length measured in centimeters.
Ac-S-Ch
AChE
Ac-COO- + Ch-S- + R-S-S-R
(DTNB)
R-S- + R-S-S-Ch
Colorimetric determination of Paraoxonase activity
PON1 efficiently catalyzes the hydrolysis of organophosphates, among them - Paraoxon,
which we will use as a substrate. Paraoxon is hydrolyzed to diethylphosphoric acid and pnitrophenol, which absorbs light at 405 nanometer (yellow color). This reaction can be
quantitatively monitored by spectrophotometric absorption (6).
Determination of Arylesterase activity
PON1 is able to hydrolize arylester bonds such as in the phenylacetate molecule.
Phenylacetate is hydrolyzed to acetic acid and phenol, which possesses an intrinsic
fluorescence. We will fluorescently monitor phenol accumulation at 280 nanometer (4).
PON
+
+ H2O
Phenylacetate
Acetic acid
Phenol
References
1. Herbst, R. S., Freidman, N., Darnell, J. E., Babiss, L. E. (1989) Positive and negative
elements in the mouse albumin enhancer. Proc Natl Acad Sci USA 86, 1553-7
2. Gott, P., Beck, S., Machado, J. C., Carneiro, F., Schmitt, H., Blin, N. (1996) Human
trefoil peptides: genomic structure in 21q22.3 and coordinated expression. Eur J Hum
Genet 4, 308-15
3. Shapira, M., Tur-Kaspa, I., Bosgraaf, L., Livni, N., Grant, A. D., Grisaru, D., Korner,
M., Ebstein, R. P., and Soreq, H. (2000). A transcription-activating polymorphism in
the ACHE promoter associated with acute sensitivity to anti-acetylcholinesterases.
Hum Mol Genet 9, 1273-1281.
4. Brophy V.H, Jampsa R.L, Clendenning J.B, McKinstry L.A, Jarvik G.P and Furlong
CE. Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1)
expression.
Am J Hum Genet. 2001 Jun;68(6):1428-36.