Beefing up an enzyme lab: Acetylcholinesterase activity in extracts from bean beetles standardized for protein content

Hector Fermin, Avajé Jackson, and Fardad Firooznia City College of New York, New York, NY 10031

Procedure for crude protein extraction The AChE enzyme assay

The Bradford assay for protein concentration

References •  Beck, C. W., and Blumer, L. S. (2011). A handbook on bean beetles,

Callosobruchus maculatus. www.beanbeetles.org. •  Bradford, M. M. (1976). A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.

•  Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7: 88-95.

•  Ffrench-Constant, R. H., and Bonning, B. C. (1989). Rapid microtitre plate test distinguishes insecticide resistant acetylcholinesterase genotypes in the mosquitoes Anopheles albimanus, An. nigerrimus and Culex pipiens. Medical and Veterinary Entomology, 3: 9-16.

•  Gbaye, O. A., Holloway, G. J., and Callaghan, A. (2012). Variation in the sensitivity of Callosobruchus (Coleoptera: Bruchidae) acetylcholinesterase to the organophosphate insecticide malaoxon: effect of species, geographical strain and food type. Pest Management Science, 68: 1265-1271.

•  Guo, F., Lei, J., Sun, Y., Chi, YH., Ge, F., Patil, BS., Koiwa, H., Zeng, R., Zhu-Salzman, K. (2012). Antagonistic regulation, yet synergistic defense: effect of bergapten and protease inhibitor on development of cowpea bruchid Callosobruchus maculatus. PLoS ONE, 7(8): e41877. doi:10.1371/journal.pone.0041877.

•  Magaña, C., Hernandez-Crespo, P., Brun-Barale, A., Couso-Ferrer, F., Bride, J-M., Castanera, P., Feyereisen, R., and Ortego, F. (2008). Mechanisms of resistance to malathion in the medfly Ceratitis capitata. Insect Biochemistry and Molecular Biology, 38: 756-762.

•  Spencer, A. G., Price, N. R., and Callaghan, A. (1998). Malathion-specific resistance in a strain of the rust red grain beetle Cryptolestes ferrugineus (Coleoptera: Cucujidae). Bulletin of Entomological Research, 88: 199-206.

Materials • Bean beetles, Callosobruchus maculatus •  Incubator with light and temperature control •  Insecticide malaoxon • ATCI and DTNB • Bradford reagent • Spectrophotometer, vortexer, centrifuge • Water bath, autoclave, micropipetters • Other standard laboratory equipment

Why use this system? • Bean beetles are common pests • Bean beetles are easy to maintain in the laboratory • Organophosphate insecticides are commonly used to control

insect pests and insects are developing resistance • Ties in several course topics and lab skills: Introduction to

enzymes, colorimetric assays, serial dilution and standard curves, the role of inhibitors in enzyme activity, cell-cell signaling, and potential ecological consequences of insecticide use

Sample Student Results

Figure 1: Standard curve for the Bradford assay.

Figure 2: Initial AChE activity measured in crude extracts of bean beetles. The values are means±SEM. Different letters indicate significant differences.

Figure 3: Relative inhibition of AChE due to malaoxon in crude extracts of bean beetles. The values are means±SEM. Different letters indicate significant

differences. Relative inhibition is calculated as % reduction in AChE activity.

Current Research

• To develop longer-term student projects to determine whether food source affects detoxification enzymes such as general esterases

• To use an artificial bean system based on Guo et al. (2012) that can be supplemented with plant defensive compounds to determine their effects on detoxification enzymes and AChE

• To isolate malaoxon-resistant bean beetle cultures and study their method of resistance to the insecticide

Acknowledgements • Biology Department, CCNY • Chris Beck, Larry Blumer, and Jennifer Smith

for beetles, instructions, beetle images, and other help • PSC-CUNY Research Award # 67161-0045

Comments •  There is variability in AChE activity and

relative inhibition of AChE due to malaoxon •  Food source affects:

! Initial AChE activity ! Sensitivity of AChE to malaoxon

•  Can be done in regular 3-hour laboratories •  Can be supplemented with readings from the

primary literature for more advanced students: ! Gbaye et al. (2012): variability in AChE activity

and sensitivity to malaoxon in different geographic strains reared on different foods

! Magaña et al. (2008): resistance due to point mutation in AChE, correlation between AChE activity and sensitivity to malaoxon

What should students know?

Acetylcholinesterase (AChE) Enzyme Assay Based on the work done by Ellman et al. (1961), Ffrench-Constant and Bonning (1989), Spencer et al. (1998), and Gbaye et al. (2012). TNB2- is yellow and can be detected spectrophotometrically. ATCI + H2O AChE Acetate + Thiocholine Iodide + 2H+

DTNB2- + Thiocholine Mixed disulfide + TNB2- (yellow)

Bradford Assay Based on Bradford (1976) to measure protein concentration in crude extracts.

• An introduction to the life cycle of the insects

• Basic structure of proteins

• Basics about enzymes • The basics of enzyme

inhibition • Basics of synaptic

signaling •  Function of

acetylcholinesterase

Image source: http://www.cnsforum.com/

educationalresources/imagebank/dementia_cholinergic/rcpt_sys_ach_esterase

used with permission

Questions or Comments: ffirooznia@ccny.cuny.edu

A A A A B B C

C

C

Beetle + Homogenize Centrifuge Transfer supernatant Centrifuge Transfer from bottom to C Vortex Centrifuge 1 mL buffer ~5 seconds 5 minutes to B, avoid pellet 5 minutes Avoid cloudy layer on top 3 seconds 10 seconds Need at least 850 µL

Mix solution

Transfer 320 µL to D and E each

Add 48 µL reaction substrate Add 200 µL DTNB

Incubate at 30°C 5 minutes

Transfer all to Cuvette

Read absorbance at 405 nm

Calculate relative inhibition

D

E

Add 320 µL Homogenizing buffer to each blank Add 48 µL reaction

substrate + malaoxon Add 200 µL DTNB

P

Transfer 200 µL to P

B

Add 200 µL Homogenizing buffer to blank Add 800 µL

Bradford dye reagent

Vortex 5 seconds

Transfer 500 µL to Cuvette

Read absorbance at 595 nm

Incubate at room temperature 10 minutes Calculate

protein concentration

y = 0.0045x - 0.0398 R² = 0.99672

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 25 50 75 100 125 150

Abs

orba

nce

(AU

)

BSA Concentration (mg/mL)

0

5

10

15

20

25

30

Mung Beans Adzuki Beans Black-eyed Peas

Initi

al A

ChE

Act

ivity

(AU

/mg

prot

ein)

Food Source

N = 13 N = 15 N = 12 a ab b

0

10

20

30

40

50

60

Mung Beans Adzuki Beans Black-eyed Peas

Rel

ativ

e In

hibi

tion

(%)

Food Source

N = 13 N = 15 N = 12 a b ab

B-M

B+M