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 µl enzyme samples into wells of a 96-well microtiter plate
3. Add 180 µl 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 µl 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 .