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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Acetyl Cholinesterase
(Acetyl choline hydrolase EC 3.1.1.7)
 
Acetyl choline esterase 1(AChE), an enzyme found in the free state mainly in brain, nerve cells, muscle, lung and erythrocytes plays an important role in the transmission of nerve impulse. The enzyme activity is strongly inhibited in cases of poisoning with organophosphorus compounds. The determination of AChE in whole blood is of importance when there is a question of possible poisoning with organophosphorus compounds. AChE is widely assayed in toxicological studies on insects. Acetyl choline is involved in phytochrome action and hence the involvement of AChE in plants for the purpose of regulating the levels of endogenous acetyl choline is suggested. Acetyl choline esterase has been reported to be present in roots, stem and leaves of pea plant and its properties have been studied.
 
 
Principle
The hydrolyzed acetyl choline per unit time is measured by comparison of the initial concentration in a reference tube with the final concentration in the experimental tube. Acetylcholine is made to react with hydroxylamine to form the corresponding acylhydroxamic acid which forms a strongly colored ferric hydroxamate with ferric salts and the color of hydroxamate is measured at 490nm.
 
 



Materials
Collect 0.2mL blood from the test animal and transfer to 5mL water. Use the haemolysate for the assay.
Veronal Buffer, 0.1M pH 8.6
   Dissolve 4.92g sodium veronal and 3.24g sodium acetate in about 300mL water, add 3mL IN HC1 and dilute to 500mL with water. Check the pH.
Acetyl Choline Stock Solution (200mM)
   Place 1.82g acetylcholine chloride (hygroscopic, check the purity) in a 50mL volumetric flask, dissolve in water and make up to volume.
Substrate Acetyl Choline Solution (1.33mM).
   Mix 150mL veronal buffer and 1mL of acetyl choline stock solution thoroughly.
Sodium Hydroxide 2.5N
   Dissolve 10g NaOH in water and make up to 100mL.
Hydroxylamine IN
   Dissolve 7g hydroxylammonium chloride in water and make up to 100mL. Store the solution in a well-stoppered polyethylene flask in a refrigerator.
Alkaline Hydroxylamine Solution
   Mix equal volumes of sodium hydroxide (2.5N) and IN hydroxylamine solutions.
Iron Solution (0.7M)
   Dissolve 33.75g Fe(NH4)(SO4)2.12 H2O in about 70mL water with gentle warming. Add 2.5g potassium nitrate (dissolved separately in water). Transfer to a 100mL volumetric flask and dilute to the mark.
Citrate Buffer 1M (pH 1.4)
    Dissolve 2.10g citric acid and 0.8g NaOH in minimum quantity of water in a 100mL volumetric flask, add 89mL 1N HCl and dilute to the mark with water. Dilute 10mL of this solution in a volumetric flask to 100mL; pH of this solution must be between 1.4 and 1.2.
    With the exception of substrate solution all other solutions are stable for several months.
Extraction of Enzyme from Plant Tissues
   Fresh sample material (root, leaf or any other part) is finely ground and extracted in 10mM veronal buffer (pH 8.6) followed by centrifugation at 20,000g for 10min. The pellet containing the enzyme is again ground and extracted with the above buffer containing 5% ammonium sulphate. The extract is centrifuged at 20,000g for 10min and the supernatant used as enzyme source.
 
 
Procedure
1.
Pipette out into 50mL volumetric flasks as given below:
 
Reference
mL
Test
mL
Blank
mL
Sample (enzyme source)
-
2
-
Substrate solution
25
25
-
Mix well and incubate at 37°C for 30 min
 
 
 
Alkaline hydroxylamine solution
5
5
5
Sample
2
-
-
Citrate buffer
5
5
5
Ferric solution
10
10
10
Allow the ferric solution to run slowly down the wall of the flask.
2.
Dilute with water to the mark and shake thoroughly.
3.
Allow to stand for 20min at room temperature.
4.
Filter the solutions through a double-folded filter paper and discard the first portion of filtrate.
5.
Measure the absorbance of the filtrates at 490nm against blank.
 
 
Calculation
The absorbance difference (AE) between reference (initial concentration of substrate) and test (final concentration of substrate) is used for calculation. For measurements at 490nm the extinction of the dye is 0.961 per micromole. Hence the amount of dye formed from the non-hydrolysed acetylcholine in 50mL
C =
E x 50
(mmoles/50mL)
0.961 x 1.0
The AChE activity in whole blood
=
E x 50
x
1
x 1000
0.961 x 1.0
0.08 x 30
                 =  E x 21667 (U/L)
 
 



Notes
1.
For assay of acetylcholinesterase in insect tissues, homogenise the tissues in veronal buffer solution, centrifuge and use aliquots of supernatant.
2.
In the case of plant extracts the enzyme activity is expressed as millimole acetylcholine hydrolyzed per hour per mg protein.
3.
The enzyme can be assayed using thiocholine esters as substrate instead of acetylcholine.
 
 
References
1.  Wolfgong Pilz (1974) In: Methods of Enzymatic Analysis (Ed Bergmeyer) Academic Press Inc New York II Edition 2 p 840.
2.  Kasturi, R and Vasantharajan, V N (1976) Phytochem 15 1345.
 
     
 
 
     




     
 
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