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.

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 todiethylphosphoric acid and p-nitrophenol, 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).

References
  1. Herbst, R.S., Freidman, N., Darnell, J.E., Babiss, L.E. (1989) Positive and negative elements in the mouse albumin enhancer.ProcNatlAcadSciUSA86, 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.