Colorimetric determination of cholinesterase activities - Ellman's assay.

Acetylcholinesterase and butyrylcholinesterase efficiently catalyze the hydrolysis of

acetyl- and butyryl-thiocholine (AcSCh and BuSCh) - sulfur analogs of their respective
natural substrate, acetylcholine. Upon hydrolysis, these substrate analogs produce acetate
(or butyrate) and thiocholine. Thiocholine in the presence of the highly reactive
dithiobisnitro-benzoate (DTNB) ion reacts to generate the yellow of 5-thio-2nitrobenzoate anion. The yellow color, can be quantified by its absorbance at 405 nm
(Ellman et al, 1961)
.
We will perform the Ellman assay in 96-well microtiter plates in a final reaction volume
of 200 l. Substrate hydrolysis is monitored by repeated spectrophotometric readings at 2
min. intervals by a computer controlled microtiter plate reader which automatically
computes mA405/min for the best fit straight line through the data points. We will then
convert our data into the standardized units of nanomoles substrate hydrolyzed/min x ml,
using the extinction coefficient for the yellow product ( = 13,600 M-1cm-1) to find the
concentration, c, from the equation c = A/xl. The light path, l, is 0.5 cm.
Lab Manual:
Protein extraction.
1. Homogenize tissue in 9 volumes of Solution D.
2. Incubate on ice for 1hr.
3. Centrifuge for 45 min at 14,000 rpm at 4 C.
4. Collect supernatant fluid to a new eppendorf tube.
Step by step through activity measurement in homogenates.
1. Prepare 50 ml Ellman's reagent without substrate (0.1 M phosphate buffer pH7.4, 0.5
mM DTNB).
2. Aliquot 10 l enzyme samples into wells of a 96-well microtiter plate
3. Add 180 l of the Ellman's reagent to each well.
4. Preincubate approximately 30 min in case you use inhibitor.
5. Prepare a 20X (20 mM) solution of substrate (acetylthiocholine iodide) and keep on ice
until used.
6. Dispense 10 l of the substrate to each well.
7. Spectrophotometric readings (405 nm) may now be taken at regular intervals using a
specially adapted microtiter plate reader; you will receive directions from the instructor
as to its operation.
Solutions and Reagents:
I. Solution D: 0.01M Tris HCl, pH 7.4, 1M NaCl, 0.01M EGTA, 1% Triton X-100.
II. Phosphate buffer 0.2 M stock (pH 7.4). Store at 4 C.
III. DTNB 15 mM stock (30x) in 0.1 M phosphate buffer (pH 7.4).
To 250 ml of Soln. II, add:
3.0 g 5,5-dithio-bis(2-nitrobenzoic acid) (Sigma D-8130).

 750 mg sodium bicarbonate (NaHCO3).

Dilute to 500 ml with DDW.
Store refrigerated in dark bottle.
IV.
Acetylthiocholine iodide (AcSCh, m.w. 289.2 g/mol) (Sigma A-5751), stored as
powder at 20 C. Make 20 mM stock by weighing 10mg of AcSCh to 1.7 ml
DDW.
V.
Ellman's reagent (slightly modified)
Final reaction concentration:
85 mM phosphate buffer.
0.425 mM DTNB.
1 mM acetylthiocholine .