Estimation of Acetylcholine Esterase | Enzyme Activity

In this article we will discuss about the tests for estimation of acetylcholine esterase in plants.

Principle:

The hydrolysed acetylcholine 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 coloured ferric hydroxamate with ferric salts and the colour of hydroxamate is measured at 490 nm.

Materials:

(i) Collect 0.2 ml blood from the test animal and transfer to 5 ml water. Use the haemolysate for the assay.

(ii) Veronal Buffer, 0.1 M pH 8.6:

Dissolve 4.92 g sodium veronal and 3.24 g sodium acetate in about 300 ml water, add 3 ml 1 N HC1 and dilute to 500 ml with water. Check the pH.

(iii) Acetyl Choline Stock Solution (200 mM):

Place 1.82 g acetycholine chloride (hygroscopic, check the purity) in a 50 ml volumetric flask, dissolve in water and make up to volume.

(iv) Substrate Acetyl Choline Solution (1.33 mM):

Mix 150 ml veronal buffer and 1 ml of acetyl choline stock solution thoroughly.

(v) Sodium Hydroxide 2.5 N:

Dissolve 10 g NaOH in water and make up to 100 ml.

(vi) Hydroxylamine 1 N:

Dissolve 7 g hydroxyl-ammonium chloride in water and make up to 100 ml. Store the solution in a well-stoppered polyethylene flask in a refrigerator.

(vii) Alkaline Hydroxylamine Solution:

Mix equal volumes of sodium hydroxide (2.5 N) and 1 N hydroxylamine solutions.

(viii) Iron Solution (0.7 M):

Dissolve 33.75 g Fe (NH₄) (SO₄)₂. 12 H₂O in about 70 ml water with gentle warming. Add 2.5 g potassium nitrate (dissolved separately in water). Transfer to a 100 ml volumetric flask and dilute to the mark.

(ix) Citrate Buffer 1 M (pH 1.4):

Dissolve 2.10 g citric acid and 0.8 g NaOH in minimum quantity of water in a 100 ml volumet­ric flask, add 89 ml 1 N HCl and dilute to the mark with water. Dilute 10 ml of this solution in a volumetric flask to 100 ml; 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.

(x) Extraction of Enzyme from Plant Tissues:

Fresh sample material (root, leaf or any other part) is finely ground and extracted in 10 mM veronal buffer (pH 8.6) followed by centrifugation at 20,000 g for 10 min. The pellet contain­ing the enzyme is again ground and extracted with above buffer containing 5% ammonium sulphate. The extract is centrifuged at 20.000 g for 10 min and the supernatant used as enzyme source.

Test Procedure:

1. The sample example and substrates etc. are pipette out into 50 ml volumetric flasks is the following manner:

The ferric solution is allowed to run slowly down the wall of the flask.

2. The mixture is diluted with water to the mark and shake thoroughly and allowed to stand for 20 min at room temperature.

3. The solutions is filtered through a double-folded filter paper and discard the first portion of filtrate.

4. The absorbance is measured of the filtrates at 490 nm 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 490 nm the extinction of the dye is 0.961 per micromole. Hence the amount of dye formed from the non-hydrolysed acetylcholine in 50 ml

C = E × 50 0.961 × 1.0 (µmol/50 ml)

The AChE activity in whole blood = E × 50/0.961 × 1.0 × 1/0.08 × 30 × 1,000 = E × 21,667 (U/L)