Ellman Esterase Assay


The assay is based on measurement of the change in absorbance at 405 nm. The method is described in detail by Ellman, G. L., et al, Biochem. Pharmacol., 7, 88-95, 1961. The assay uses the thiol ester acetylthiocholine instead of the oxy ester acetylcholine. AChE hydrolyses the acetylthiocholine to produce thiocholine and acetate. The thiocholine in turn reduces the Dithiobis-Nitrobenzoic Acid liberating nitrobenzoate, which absorbs at 405 nm. The reaction is shown below in the Figure from ESTHER.


Reagent Stock Solutions for Standard Activity Measurement

  1. Color Indicator, DTNB: 10 mM 51, 51 Dithiobis (2-Nitrobenzoic Acid) (3.96 mg/ ml) in 100 mM NaHPO4, pH 7.0-8.0, containing 1.5 mg NaH2CO4.
  2. Substrate: 75 mM Acetylthiocholine Iodide (21.67 mg/ml) or 7.5 mM Butyrylthiocholine Iodide (. mg/ml) in H2O. (Compensated for ilution in assay.)
  3. Buffer: 100 mM NaHPO4, pH 7.0-8.0.

Extraction and Measurement of Acetylcholinesterase Activity

Extraction of AChE from 75 cm2 Tissue Culture Flasks.

  1. Remove medium from the cells by aspiration.
  2. Wash the cells three times with 8 ml of Phosphate-Buffered Saline (PBS) three times. To wash cells, simply add PBS gently and then remove it by aspiration.
  3. Extract the cells by adding 4 ml of High Ionic Strength (HIS) buffer (10 mM NaHPO4, pH 7.0-8.0, 1 M NaCl, 10% Triton X-100, 1 mM EDTA) to each well. Incubate 5 minutes at room temperature. The detergent, Triton X-100, and the high NaCl concetration effectively solubilizes the cells and extracts the proteins.
  4. Centrifuge at 12,000 rpm in a 15 ml Corex tube for 20 minutes.
  5. Transfer the supernatant to an appropriate storage tube.

Extraction of AChE from Six-well Plates.

  1. Remove medium from the cells by aspiration.
  2. Wash the cells three times with 1 ml of Phosphate-Buffered Saline (PBS) three times. To wash cells, simply add PBS gently and then remove it by aspiration.
  3. Extract the cells by adding 0.5 ml of High Ionic Strength (HIS) buffer (10 mM NaHPO4, pH 7.0-8.0, 1 M NaCl, 10% Triton X-100, 1 mM EDTA) to each well. Incubate 5 minutes at room temperature. Combine the extract from appropriate wells. The detergent, Triton X-100, and the high NaCl concetration effectively solubilizes the cells and extracts the proteins.
  4. Centrifuge at 12,000 rpm in a 15 ml Corex tube for 20 minutes.
  5. Transfer the supernatant to an appropriate storage tube.

Measurement of AChE Activity

We will measure AChE activity in a 96 well microtitre plate reader, which is essentially capable of reading 96 separate enzyme assays simultaneously. To access the kinetic software program associated with the microplate reader, turn on the plate reader and click on the kc4 icon on the desk top.

Measurement of AChE Activity I: Standard Activity Measurement

The first assay that you will want to do is a simple activity measurement to see if the AChE was expressed by the cells.

  1. Mix Buffer, Substrate, and DTNB in a ratio of 150:2:5 to give a final concentration of acetylthiocholine of 1 mM and butyrylthiocholine of 0.1 mM. Examples are shown in the Table below. You need 300 ul of Ellman solution for each activity measurement. Make 15 mls recipe.

Sodium Phosphate Buffer

Substrate: AsCh or BsCh

Color Reagent: DTNB

150 ml

2 ml

5 ml

15 ml

200 ul

500 ul

30 ml

400 ul

1 ml

45 ml

600 ul

1.5 ml

  1. We will measure AChE activity in a 96 well microtitre plate reader, which is essentially capable of reading 96 separate enzyme assays simultaneously. To access the kinetic software program associated with the microplate reader, click on the kc4 icon on the desk top. Select New, Protocol, Open, AChE1, ache1.prt, and Read. Enter your Name in Prompt 1, the name of the Experiment in Prompt 2, and a Description of the Experiment where indicated.
  2. Transfer 50 ul of extract from transfected cells to a well in a 96 well microtitre dish. Do not transfer the first pipet-full that you take, but pipet that back into the solution. Since the buffer contains detergent, it is good practice to prewet the tip in this way. Then pipette as usual. The matrix below correspnds to the layout of a microtitre plate. Pipette 50 ul of extract from untransfected cells to serve as a negative control in the well below. Position the micropipettor so the tip is touching the side of the well and the solution runs down the side. This technique will give you a more reproducible volume. Pop the large bubbles that will form due to the detergent that is present in the buffer. The technique will be demonstrated.
  3. Pipette 300 ul of Ellman Assay Solution into the two rows below. For example, for six samples, assaying each flask in triplicate:
 

1

2

3

4

5

6

7

8

9

10

11

12

A AChE-A AChE-A AChE-A AChE-B AChE-B AChE-B            
B Control Control Control Control Control Control            
C Ellmans Ellmans Ellmans Ellmans Ellmans Ellmans            
D Ellmans Ellmans Ellmans Ellmans Ellmans Ellmans            
E                        
F                        
G                        
H                        
  1. Use a multichannel pipette to transfer 250 ul of the Ellmans Assay Solution to the Control and Experimental wells. Note: Do not push the plunger to the second stop when adding the assay solution to the sample wells; this will cause bubbles to reform.
  2. Immediately place the plate in the reader and press Read Sample. The computer will show the change in absorbance as a function of time for each well and will calculate the rate in mOD/min and the regression coefficient for goodness of fit. All of these data will be printed out in a report at the end of the experiment. Let the assay proceed for 3-10 minutes.
  3. At the end of the assay, a report will print automatically. For multiple reports, select Print. Save your file under the name of your experiment in your own folder. Do not overwrite a file if someone else has chosen the same file name.
  4. The negative controls are needed because thiol esters are unstable compared to oxy esters and there will be some spontaneous hydrolysis of the substrates. This spontaneous breakdown must be subtracted from the experimental rate to get the true enzyme-catalyzed rate, and we will do this manually by subtracting each control value from its corresponding experimental value above it.
Measurement of AChE Activity II: Kinetic Constants for Acetylthiocholine and Butyrylthiocholine

In this experiment, you will determine Km, Kss, and Vmax for acetylthiocholine and butyrylthiocholine. The assay will again make use of the Ellman Solution and microtitre plate assay, but the microtitre plate will be configured in a different way and serial dilutions of both substrates will be included in separate assays. Note: You will perform this measurement of kinetic constants twice, once for acetylthiocholine and once for butyrylthiocholine.

  1. We will measure AChE activity in a 96 well microtitre plate reader, which is essentially capable of reading 96 separate enzyme assays simultaneously. To access the kinetic software program associated with the microplate reader, click on the kc4 icon on the desk top. Select New, Protocol, Open, AChE1, ache1.prt, and Read. Enter Your Name in Prompt 1, the name of the Experiment in Prompt 2, and a Description of the Experiment where indicated.
  2. Pipette 50 ul of experimental AChE extract into the wells in lane A and 50 ul of control extract into the wells of lane B.
 

1

2

3

4

5

6

7

8 9 10 11 12
A AChE

1 X 10-7

Substrate

AChE

3.16X10-7

Substrate

AChE

1 X 10-6

Substrate

AChE

3.16X10-6

Substrate

AChE

1 X 10-5

Substrate

AChE

3.16X10-5

Substrate

AChE

1 X 10-4

Substrate

AChE

3.16X10-4

Substrate

AChE

1 X 10-3

Substrate

AChE

3.16X10-3

Substrate

AChE

1 X 10-2

Substrate

AChE

3.16X10-2

Substrate

B Control

1 X 10-7

Substrate

Control

3.16X10-7

Substrate

Control

1 X 10-6

Substrate

Control

3.16X10-6

Substrate

Control

1 X 10-5

Substrate

Control

3.16X10-5

Substrate

Control

1 X 10-4

Substrate

Control

3.16X10-4

Substrate

Control

1 X 10-3

Substrate

Control

3.16X10-3

Substrate

Control

1 X 10-2

Substrate

Control

3.16X10-2

Substrate

C Ellman's

1 X 10-7

Ellman's

3.16X10-7

Ellman's

1 X 10-6

Ellman's

3.16X10-6

Ellman's

1 X 10-5

Ellman's

3.16X10-5

Ellman's

1 X 10-4

Ellman's

3.16X10-4

Ellman's

1 X 10-3

Ellman's

3.16X10-3

Ellman's

1 X 10-2

Ellman's

1 X 10-2

D Ellman's

1 X 10-7

Ellman's

3.16X10-7

Ellman's

1 X 10-6

Ellman's

3.16X10-6

Ellman's

1 X 10-5

Ellman's

3.16X10-5

Ellman's

1 X 10-4

Ellman's

3.16X10-4

Ellman's

1 X 10-3

Ellman's

3.16X10-3

Ellman's

1 X 10-2

Ellman's

3.16X10-2

E                        
F                        
G                        
H                        
  1. Then prepare Ellman Solution without substrate. Make the 45 ml recipe:

Sodium Phosphate Buffer

Color Reagent: DTNB

150 ml

5 ml

15 ml

500 ul

30 ml

1 ml

45 ml

1.5 ml

  1. Next prepare 13 test tubes labeled as shown below:
 

1

2

3

4

5

6

7

8 9 10 11 12 13
A 1 X 10-7

Substrate

3.16X10-7

Substrate

1 X 10-6

Substrate

3.16X10-6

Substrate

1 X 10-5

Substrate

3.16X10-5

Substrate

1 X 10-4

Substrate

3.16X10-4

Substrate

1 X 10-3

Substrate

3.16X10-3

Substrate

1 X 10-2

Substrate

3.16X10-2

Substrate

1 X 10-1

Substrate

B 1368 ml

Ellman's

1.368 ml

Ellman's

1368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

1.368 ml

Ellman's

2 ml

Ellman's

                          70 mg of AsCh or

76 mg of BsCh

    <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul <632 ul
  1. Weigh out either the 70 mg of AsCh or the 76 mg of BsCh and add it to the 2 ml of Ellman's solution in tube 1. Vortex until it has dissolved completely. It is more difficult to dissolve the BsCh. As soon as it is dissolved, transfer 632 ul of the substrate solution from tube 1 to tube 2. Rinse the pipet in the solution in tube 2 and then vortex tube 2. Repeat these steps of transfer, rinsing, and vortexing for each tube finishing up by vortexing tube 8.
  2. Transfer 300 ul of Ellman's with each concentration of substrate into the appropriate well of lanes C and D. Do not transfer tube 13.
  3. Use a multichannel pipette to transfer 250 ul of Ellman's solution in C and D to the corresponding wells in A and B.
Measurement of AChE Activity III: Dose-Response Curve for Four Pharmacological Inhibitors

In this experiment you will determine the sensitivity of the enzyme to four inhibitors. The assay will again make use of the Ellman Solution and microtitre plate assay, but the microtitre plate will be configured in a different way, only AsCh will be used, and a serial dilution of drug will be included in the assay. Note: You will perform this measurement of drug sensitivity twice, each time with two different drugs, for a total of four drugs: BW284c51, Ethopropazine, Eserine, and Propidium.

  1. Mix Buffer, Substrate, and DTNB in a ratio of 150:2:5 to give a final concentration of acetylthiocholine of 1 mM and butyrylthiocholine of 0.1 mM. Examples are shown in the Table below. Make the 45 ml recipe.

Sodium Phosphate Buffer

Substrate: AsCh or BsCh

Color Reagent: DTNB

150 ml

2 ml

5 ml

15 ml

200 ul

500 ul

30 ml

400 ul

1 ml

45 ml

600 ul

1.5 ml

  1. We will measure AChE activity in a 96 well microtitre plate reader, which is essentially capable of reading 96 separate enzyme assays simultaneously. To access the kinetic software program associated with the microplate reader, click on the kc4 icon on the desk top. Select New, Protocol, Open, AChE1, ache1.prt, and Read. Enter Your Name in Prompt 1, the name of the Experiment in Prompt 2, and a Description of the Experiment where indicated.
  2. Pipette 5 ul of water or indicated concentration of drug 1 into rows A and B, and drug 2 into rows C and D.
  3. Pipette 25 ul of water or indicated concentration of drug 1 into rows E and F, and drug 2 into rows G and H.
  4. Transfer 45 ul of extract from transfected cells to 8-12 wells, depending on the drug, in rows A and C in a 96 well microtitre dish. The matrix below corresponds to the layout of a microtitre plate. Pipette 45 ul of extract from untransfected cells to 8-12 wells, depending on the drug, in rows B and D, to serve as a negative control. Position the micropipettor so the tip is touching the side of the well and the solution runs down the side. This technique will give you a more reproducible volume. Pop the large bubbles that will form due to the detergent that is present in the buffer. The technique will be demonstrated.
  5. Pipette 225 ul of AsCh-Ellmans Assay Solution into rows E, F, G, and H. For example see the template below.
  6. Note: the concentration of the drug used will depend on the drug. In some cases low concentrations of drug will be used. In other cases high concentrations will be used. The template is simply an example. Also note that the final concentration of each drug in the assay is 10X lower than the stock solution used because of the dilution of the drug in the extract and in the assay solution. So, 1 X 10-2 becomes 1 X 10-3.
 

1

2

3

4

5

6

7

8

9

10

11

12

A AChE

water

AChE

1 X 10-7

AChE

3 X 10-7

AChE

1 X 10-6

AChE

3 X 10-6

AChE

1 X 10-5

AChE

3 X 10-5

AChE

1 X 10-4

AChE

3 X 10-4

AChE

1 X 10-3

AChE

3 X 10-3

AChE

1 X 10-2

B Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

C AChE

water

AChE

1 X 10-7

AChE

3 X 10-7

AChE

1 X 10-6

AChE

3 X 10-6

AChE

1 X 10-5

AChE

3 X 10-5

AChE

1 X 10-4

AChE

3 X 10-4

AChE

1 X 10-3

AChE

3 X 10-3

AChE

1 X 10-2

D Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

Control

water

E AsCh-Ell.

water

AsCh-Ell.

1 X 10-7

AsCh-Ell.

3 X 10-7

AsCh-Ell.

1 X 10-6

AsCh-Ell.

3 X 10-6

AsCh-Ell.

1 X 10-5

AsCh-Ell.

3 X 10-5

AsCh-Ell.

1 X 10-4

AsCh-Ell.

3 X 10-4

AsCh-Ell.

1 X 10-3

AsCh-Ell.

3 X 10-3

AsCh-Ell.

1 X 10-2

F AsCh-Ell.

water

AsCh-Ell.

1 X 10-7

AsCh-Ell.

3 X 10-7

AsCh-Ell.

1 X 10-6

AsCh-Ell.

3 X 10-6

AsCh-Ell.

1 X 10-5

AsCh-Ell.

3 X 10-5

AsCh-Ell.

1 X 10-4

AsCh-Ell.

3 X 10-4

AsCh-Ell.

1 X 10-3

AsCh-Ell.

3 X 10-3

AsCh-Ell.

1 X 10-2

G AsCh-Ell.

water

AsCh-Ell.

1 X 10-7

AsCh-Ell.

3 X 10-7

AsCh-Ell.

1 X 10-6

AsCh-Ell.

3 X 10-6

AsCh-Ell.

1 X 10-5

AsCh-Ell.

3 X 10-5

AsCh-Ell.

1 X 10-4

AsCh-Ell.

3 X 10-4

AsCh-Ell.

1 X 10-3

AsCh-Ell.

3 X 10-3

AsCh-Ell.

1 X 10-2

H AsCh-Ell.

water

AsCh-Ell.

1 X 10-7

AsCh-Ell.

3 X 10-7

AsCh-Ell.

1 X 10-6

AsCh-Ell.

3 X 10-6

AsCh-Ell.

1 X 10-5

AsCh-Ell.

3 X 10-5

AsCh-Ell.

1 X 10-4

AsCh-Ell.

3 X 10-4

AsCh-Ell.

1 X 10-3

AsCh-Ell.

3 X 10-3

AsCh-Ell.

1 X 10-2

  1. Use a multichannel pipette to transfer 200 ul of the AsCh-Ellmans Assay Solution in H, G, F, and E to the Experimental and Control wells D, C, B, and A. Note 1: Transfer the Ellmans Solution in the order given. Note 2: Do not push the plunger to the second stop when adding the assay solution to the sample wells; this will cause bubbles to reform.
  2. Immediately place the plate in the reader and press Read Sample. The computer will show the change in absorbance as a function of time for each well and will calculate the rate in mOD/min and the regression coefficient for goodness of fit. All of these data will be printed out in a report at the end of the experiment.
  3. At the end of the assay, a report will print automatically. For multiple reports, select Print. Save your file under the name of your experiment in your own folder. Do not overwrite a file if someone else has chosen the same file name.
  4. The negative controls are needed because thiol esters are unstable compared to oxy esters and there will be some spontaneous hydrolysis of the substrates. This spontaneous breakdown must be subtracted from the experimental rate to get the true enzyme-catalyzed rate, and we will do this manually by subtracting each control value from its corresponding experimental value above it.
  5. These dose-response curves will be plotted as semi-log graphs in Sigma Plot.

Data Analysis

We will use the program SigmaPlot to help us analyze the data collected.

Michaelis-Menten Kinetic Experiments

  1. For kinetic experiments, enter the data in the following way. Label the columns by double-clicking on the column number.
  [Substrate] mAb/min AsCh mAb/min BsCh 4 5 6 7
1 1e-7 0.035 0.35        
2 3.16e-7 0.041 0.41        
3 1e-6 0.045 0.436        
4 3.16e-6 0.048 1.83        
5 1e-5 2.93 6.73        
6 3.16e-5 7.18 18.62        
7 1e-4 28.49 50.71        
8 3.16e-4 66.99 61.39        
9 1e-3 173.85 29.21        
10 3.16e-3 214.33 15.09        
11 1e-2 197.10 11.84        
12 3.16e-2 156.67 7.76        
             
  1. Click on the scatter plot icon (box with dots in it) at the top left of the screen to create a scatter plot, and selected the same icon from the submenu.
  2. A popup menu will appear with the option XY pair selected. Click on next.
  3. Select [Substrate] for Data for X by selecting that column with the mouse arrow.
  4. Select mAb/min AsCh for Data for Y.
  5. Click on finish.
  6. Click on the yellow ruler icon on the right hand side of the screen to adjust the x axis to a log scale.
  7. In the popup menu, change Axis to X data, and change Scale Type to Log (Common). Click on OK.
  8. Double click on X Data on the graph and change the label to [Substrate] Molar. Click on OK.
  9. Double click on Y Data on the graph and change the label to Cholinesterase Activity. Click on OK.
  10. Click on 2D Graph 1 and delete.
  11. Click on Graph at the top of the screen and select Add Plot from the drop down menu.
  12. Scatter Plot is selected. Click on Next. Simple Scatter is selected. Click on Next. XY Pair is selected. Click on Next.
  13. Select [Subtrate] for Data for X and mAb/min BsCh for Data for Y.
  14. Click on Finish.
  15. Double click on the new plot that appears and change the Fill Color to white in the popup menu.
  16. Double click on the curve to fit the AsCh data to the equation:

which is based on the following scheme where "S combines at two discrete sites, forming two binary complexes, ES and SE, only one of which, ES, results in substrate hydrolysis. We have assumed for simplification, that S combines equally well with E and ES. The parameter, b, reflects the efficiency of hydrolysis of the ternary comlex, SES, relative to ES." If b is zero, then the SES complex is inactive; if b < 1, then the SES complex is less active than ES; in both these cases substrate inhibition occurs. If b = 1, then the SES complex is as active as ES; if b > 1, then the SES complex is more active than ES, and the phenonemon of substrate activation occurs. Zoran Radic, Natitlie A. Pickering, Daniel C. Vellom, Shelley Camp, and Palmer Taylor. Three Distinct Domains in the Cholinesterase Molecule Confer Selectivity for Acetyl- and Butyrylcholinesterase Inhibitors. Biochemistry 1993, 32, 12074-12084. The kinetics of AChE is more complex than traditional Michaelis-Menten kinetics because of the presence of a secondary binding site for substrate and the phenomenon of substrate inhibition. Kss is the dissociation constant for the binding of substrate to the second site.

Click on Statistics at the toolbar at the top of the screen and then select Regression Wizard from the drop down menu. Select Hyperbola as the Equation Category. Select Taylor Fit as the equation. Click on Finish. Look at the graph. It should have a line fit to the points. Repeat for BsCh. To obtain Vmax, Km, Kss, and b look at the Data Page. For each fit: Vmax, Km, Kss, and b will be in rows 1, 2, 3, and 4 of a column just past the data entry columns. Record these values.

  1. Click on the box covering the X axis label and delete.
  2. Save everything in your file.

Dose-response Curves

  1. For dose-response experiments, enter the data in the following way. Label the columns by double-clicking on the column number.
  [Drug] mAb/min AsCh mAb/min BsCh 4 5 6
1 1e-7 82.5 30.3      
2 3.16e-7 81.5 31.1      
3 1e-6 82.5 30.8      
4 3.16e-6 78.3 28.5      
5 1e-5 60.2 21.3      
6 3.16e-5 45.6 17.1      
7 1e-4 30.9 11.4      
8 3.16e-4 10.7 3.9      
9 1e-3 5.0 1.6      
10 3.16e-3 1.6 0.6      
11 1e-2 0.2 0      
12            
             
  1. Click on the scatter plot icon (box with dots in it) at the top left of the screen to create a scatter plot, and selected the same icon from the submenu.
  2. A popup menu will appear with the option XY pair selected. Click on next.
  3. Select [Drug] for Data for X by selecting that column with the mouse arrow.
  4. Select mAb/min AsCh for Data for Y.
  5. Click on finish.
  6. Click on the yellow ruler icon on the right hand side of the screen to adjust the x axis to a log scale.
  7. In the popup menu, change Axis to X data, and change Scale Type to Log (Common). Click on OK.
  8. Double click on X Data on the graph and change the label to [Drug] Molar. Click on OK.
  9. Double click on Y Data on the graph and change the label to Cholinesterase Activity. Click on OK.
  10. Click on 2D Graph 1 and delete.
  11. Click on Graph at the top of the screen and select Add Plot from the drop down menu.
  12. Scatter Plot is selected. Click on Next. Simple Scatter is selected. Click on Next. XY Pair is selected. Click on Next.
  13. Select [Drug] for Data for X and mAb/min BsCh for Data for Y.
  14. Click on Finish.
  15. Double click on the new plot that appears and change the Fill Color to white in the popup menu.
  16. Double click on the curve to fit the AsCh data to the equation:
  17. Click on Statistics at the toolbar at the top of the screen and then select Regression Wizard from the drop down menu.
  18. Select Sigmoidal in the Equation Category, then Logistic, 3 parameter as the equation. Click on Finish. Look at the graph. It should have a line fit to the points. Repeat for BsCh.
  19. Save everything in your file.

The next step is to normalize the data to % Control Acetylcholinesterase Activity. To perform this operation, all of the data must be divided by the maximum value (100%). This value is obtained from the curve fit. To view the maximum value, go to the data page and note the values in 4-1 and 9-1, 85.49 and 32.43, which represent the maximum values for AsCh and BsCh hydrolysis.

Next, select Transforms, enter col(15)=(col(2)/85.49)*100, and select Execute.

Select Transforms again, enter col(16)=(col(3)/32.43)*100, and select Execute.

Now delete the current plots by double clicking on the points and lines and hitting delete. Do not delete the graph itself.

Plot the normalized data in Columns 15 and 16 against [Drug]. Change the color of the BsCh symbols to white. Fit the curves.

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