This document summarizes research testing cholinesterase activity in the blood and brain cells of Tritonia diomedea sea slugs. It describes background on cholinesterase and its role in the nervous system. Methods included colorimetric assays using acetylthiocholine iodide and butyrylthiocholine iodide substrates on sea slug blood and eel acetylcholinesterase. Results showed cholinesterase activity increased with greater substrate and blood volumes. Further testing will examine brain cell cholinesterase activity to better understand the enzyme's function in sea slug nervous systems.

1. Research Director: Dr. Monika Sommerhalter
Presentation by:
Mary Grace Villanueva & Summervir Cheema

2. Objective
 To test Cholinesterase activity in Tritonia diomedea
(sea slug) blood and brain cells

3. Background
 Cholinesterase is an important enzyme involved in
the function of the nervous system and inhibits
chemicals that are toxic
cholinesterase
(CH3)3N+CH2CH2O-OCCH3  (CH3)+NCH2CH2OH
+ +
H2O CH3COOH
(acetylcholine + water) (choline + acetic acid)

4. Background
 Acetylcholine (ACh) is a neutrotransmitter found in
nerve, muscle, blood, and other tissues that degrades
through hydrolytic activity by acetylcholinesterase
(AChE) producing choline and acetate group.
 High catalytic activity
 Butyrylcholine (BCh) is a neutrotransmitter found in
blood plasma

5. Materials Glutathione (1 mM)
 Prepared fresh
 DTNB color reagent (10.3 mM)
 Store up to 1 month @ -20°C
 ChE assay buffer
 0.1 M sodium phosphate, pH 8.0
 0.1 M sodium phosphate, pH 7.0
 Precaution: store up to 6 months @ room temperature
 ATCh substrate (10.7 mM) - S-acetylcholide iodine
 Store up to 1 month @ -20°C (but fresh solution is recommended)
 BTCh substrate (10 mM) – S-butyrylthiocholine iodine
 Store @ -20°C
 Enzyme (100 U/ml)
 1000 U eel AChE – Sigma-Aldrich cat. No C2888
1.53 mg solid; 658 units/mg solid; 1210 units/mg protein
store @ -20°C
 Tritonia diomedea – store @ -80°C

6. Methods
 Glutathione standard curve
 Colormetric Assay
 Cholinesterase Activity Assay
 Used different amount of ATChE substrate (both
concentrated and diluted)
 Enzyme Activity Assay
 Used different amount of slug blood
 BCA test

7. Procedure
 Preparation of Glutathione
 Add reagents
 1 mM glutathione, ChE assay
buffer, 10.3 mM DTNB, ATCh
substrate
 Incubate 5-15 mins at room
temperature
 Measure absorbance @ 412nm

8. Procedure
 Colormetric Assay Volumes
 Added different reagents
 ChE assay buffer, DTNB color reagent, ChE, ATCh substrate
 Controls:
 Blank without substrate
 Measure how much –SH groups were cleaved
 Blank without enzyme
 Measure activity of substrate alone
 Sample (1:100 ChE dilution) – contained all reagents

9. Procedure
 Cholinesterase Activity Assay
 Prepared 12 different assays using various amounts of
ATChE substrate for both undiluted and 1:10 dilution
 ATCh substrate: weigh out 0.03 g of S-acetylcholide iodine
and dissolved in 2ml volumetric flask
 Measured 200 µl out of the 2ml volumetric flask and
transferred it into another 2 ml volumetric flask for 1:10
dilution
 Total volume = 3200 µl

10. Procedure
 Enzyme Activity Assay
 Prepared test tubes for both ChE and slug blood
 Sample
 No substrate
 No enzyme
 Absorance @ 412nm
 Compared results of ChE vs slug blood activity

11. Procedure
 BCA test
 Prepared BSA standard curve
according to protocol
 Working Reagent (WR) = 100 ml
reagent A + 2 ml reagent B
 Mixed 3 ml WR + 0.15 ml sample
 Incubate @ 37°C in water bath
for 30 mins
 Absorbance @ 562 nm

12. Results/Discussion
Glutathione Standard Curve:
Final volume:3200 microliter/tube
 Slope: 0.0059

13. Glutathione Standard Curve
y = 0.0059x
R² = 0.9867
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 20 40 60 80 100 120 140 160
Absorbanceat412nm
nmol SH
Series1
Series2
Linear (Series2)

14. Glutathione Standard Curve
y = 0.0058x - 0.0603
R² = 0.9693
y = 0.0046x - 0.013
R² = 0.9957
y = 0.0068x - 0.1069
R² = 0.9489
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 20 40 60 80 100 120 140 160 180 200
Absorbance@412nm
nmol -SH
Series1
Series2
Series3
Linear (Series1)
Linear (Series2)
Linear (Series3)

15. Results/ Discussion
 As the nmols of Thiol group increase, the absorbance
increases as well.
 Glutathione was used in the absence of the enzyme 
activity occurred

16. Results/Discussion
 Colormetric Assay
 No substrate in the blank = no activity at 412 nm.
 No enzyme in the blank = very slight activity at 412 nm.
 Sample without dilution did not work, there was too
much enzyme activity (1.999)
 Sample with (1:100) dilution worked best with 20 sec
time difference.

17. Trial 1:
y = 0.0302x + 0.1177
R² = 0.998
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 2 4 6 8 10 12
Absat412nm
Time (min)
ChE sample Reading (1:100) dilution
Series1
Linear (Series1)

18. Trial 2:
y = 0.0014x + 0.1225
R² = 0.9941
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 50 100 150 200 250
ChE sample reading (1:100)dilution
Series1
Linear (Series1)

19. ( Trial1+Trial 2)
y = 0.0857x + 0.1225
R² = 0.9941
y = 0.0906x + 0.1177
R² = 0.998
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 0.5 1 1.5 2 2.5 3 3.5
Absorbanceat412nm
time (min)
ChE samle reading (1:100) dilution
Series3
Series4
Series1
Series2
Linear (Series1)
Linear (Series2)

20.  More trials gives you best linear relationship of slope.
 Activity of ChE in colorometric assay: 64.17891
 To calculate the activity following formula was used:
 Activity=[(average A-Ablank)/min]/factor(A/-
SHnmol)/sample vol(ml)/sample
dilution/1000(nmol/micromol)

21. Results/Discussion
 Cholinesterase activity assay
 We found out variation in slope by using different
concentrations of ATChE.
 Slope fluctuates ( increase & decrease) by using
different concentrations of ATChE.

22. Results/Discussion
0
0.01
0.02
0.03
0.04
0.05
0.06
0 0.5 1 1.5 2 2.5
slope(Abs/min)
ATChe concentration in mM
Variation in Slope
Series1

23. Results/Discussion
 Enzyme Activity Assay
 cholinesterase activity in slug blood
 Found out that when there is no substrate present in the
blank there is very slight activity.
 And same with the enzyme. When there is no enzyme
present in the blank there is very less activity.
 Found out that blank containing only slug blood
generated large increase in activity.
 Found out that activity increases as you increase the
volume of slug blood.
 Increase in slope= Inc in Activity

24. Results/Discussion
y = 0.0002x + 0.1064
R² = 0.9981
y = 0.0004x + 0.1166
R² = 0.9993
y = 0.0005x + 0.1211
R² = 0.9995
0
0.05
0.1
0.15
0.2
0.25
0.3
0 50 100 150 200 250 300
Absorbanceat412nm
Time (sec)
ChE activity in Slug blood
30 ul slug blood
60 ul slug blood
90 ul slug blood

25. Results/Discussion
y = 6E-06x
R² = 0.997
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
0 10 20 30 40 50 60 70 80 90 100
slope(abs412/sec)
ul slug blood in 3200ul
Slope Vs. Volume

26. Results/Discussion
 BCA test
 Found out that as the BSA(protein) concentration
increase the absorbance increases and vice Versa.
 Inc in abs =Inc in activity.
 Activity in protein= 528 +/- 18 µg/ml
 slope 0.001373

27. y = 1.400E-03x
R² = 9.963E-01
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 200 400 600 800 1000 1200
Absat562nm
BSA µg/ml
BCA assay May 18,2010

28. Conclusion:
 In our assays we measured volumes in micro liters.
 Our assumption is that we would get more accurate
results by using large quantities of volumes of different
reagents.
 Also our next task is to find out the activity of
Cholinesterase in Brain cells. It works the same as the
BCA assay.

29. References
 Butyrylcholinesterase: Structure and Physiological
Importance
 http://www.turkjbiochem.com/2003/054_061.pdf