Biochemical estimation of AChE (Ellman method)

Principle: Thiocholine released from acetylthiocholine by the AChE, reacts with

the 5, 5’-dithiobis-(2-nitrobenzoic acid) (DTNB), reducing it to the thiol which has an
adsorption maximum at 412 nm. An organophosphorus anticholinesterase which is a
selective inhibitor for BChE is added to suppress the potential contribution of the later to
the hydrolysis of acetylthiocholine.

Reagents:

(a) 0.1M Na/K phosphate buffer, pH 7.9

(b) 0.1M Na/K phosphate buffer, pH 7.2

(c) Tetraisopropylpyrophosphoramide (0.1mM in ‘a’)

(d) DTNB, 39.6 mg/10ml of b + 15 mg of sodium bicarbonate; can be used for 2

weeks at 4оC.

(e) Acetylthiocholine iodide, 0.075 M (21.67mg/ml); it can be kept for 10-15 days at
4оC.

Procedure:

• To enzyme, reagent c was added to make up to 3 ml; incubated at 37 оC for 30

minutes.

• Solution was cooled to room temperature, placed in spectrophotometer cell, and

0.1 ml of d and 20μl of e was added. Rate of change of adsorption was
measured at 412 nm.

Biochemical estimation of BChE (Ellman method)

Principle: Thiocholine released from butyrylthiocholine by the enzyme reacts

with 5, 5’-dithiobis-(2-nitrobenzoic acid) (DTNB), reducing it to the thiol which has
maximum absorption at 412 nm. A selective inhibitor for AChE is added to suppress the
potential contribution of the later to the hydrolysis of butyrylthiocholine.

Reagents:

(a) 0.1M Na/K phosphate buffer, pH 7.9

(b) 0.1M Na/K phosphate buffer, pH 7.2

(c) BW284 dibromide, 2.83 mg/50 ml of a (100μM)

(d) DTNB, 39.6 mg/10ml of b + 15 mg of sodium bicarbonate; it can be used for 2

weeks at 4оC

(e) Butyrylthiocholine iodide; 24 mg/ml; it can be kept for 10-15 days at 4 оC.

Procedure:

• To enzyme, reagent c was added to make up to 3 ml; incubated at 37 оC for 30

minutes.

• Solution was cooled to room temperature, placed in spectrophotometer cell, and

0.1 ml of d and 20μl of e was added. Rate of change of adsorption was
measured at 412 nm.

Biochemical estimation of ATPase (Hosie method)

Principle: Inorganic phosphate splitted off from ATP is measured by means of
the phosphomolybdate-stannous chloride method under conditions which do not favour
nonenzymic breakdown of ATP. ATP hydrolysis in tubes containing Na+, K+ (solution d
and e) and enzyme is compared with similar tubes from which Na+ and K+ have been
omitted.

Reagents:

(a) Tris-HCl buffer, 0.4M, pH 7.4

(b) EDTA-Tris, 0.02M

(c) MgCl2, 0.2M

(d) NaCl, 2M

(e) KCl, 0.2M

(f) Dinitrophenol, 0.002M

(g) CaCl2, 0.2M

(h) ATP, 0.06M (fresh for each experiment)

(i) Trichloroacetic acid, 22.5% w/v

(j) Ammonium molybdate, 10% w/v

(k) H2SO4, 2.5N

(l) Isobutyl alcohol : petroleum ether (4:1)

(m) H2SO4, 1.0N in ethanol

(n) SnCl2, 0.05% in 2.5 N H2SO4 (prepared fresh for each experiment)

Procedure:

• Enzyme sample was diluted and preincubated at 37оC for 5-10 minutes before
use.

• To sorvall glass centrifuge tube, added 0.8 ml of reagent a, 0.1ml of reagent b

and 0.1 ml of reagent h. Then following solutions were added in given order: 0.03
ml of c, 0.15 ml of d, 0.2 ml of e, 0.1 ml of f and 0.03 ml of g.

• Volume was made up to 6.9ml with distilled water and placed in a 37оC water
bath for 5 minutes.

• Added 0.1 ml of preincubated enzyme and left for exactly 10 minutes.

• For blanks, tubes were prepared containing no enzyme and containing enzyme
with no ATP mixture.

• After 10 minutes 0.5 ml of reagent i was added and placed in ice bath for 15
minutes.

• Centrifuged for 10 minutes at 5000 rpm.

• Removed 0.8 ml of supernatant to test tube in ice and added 0.2 ml of reagent k,
mixed well.

• Added 0.2 ml of j; mixed.

• Added 2.0 ml of l and stir on vortex mixer 15 seconds

• Left at room temperature for 15 minutes.

• Removed 1.4 ml of organic layer (upper) and added 1.4 ml of M; mixed.

• Added 0.2 ml of n and mixed. Tubes were left for 15 minutes and absorbance
was read at 650 nm.

Histochemical method for AChE

Direct colouring method described by Karnovsky and Roots (1964) was adapted
for the demonstration of AChE.

Incubation medium:
5 mg of substrate (i.e. acetylthiocholine iodide) was dissolved in 6.5 ml of 0.1M
phosphate buffer (pH = 6.0). Then following items were added in order while stirring-

(1) Tri sodium citrate 0.1M = 0.5 ml

(2) Copper sulfate 0.03M = 1 ml

(3) Distilled water = 1 ml

(4) Potassium ferricyanide 0.005M = 1 ml

Procedure:

• 15 μm thick sections were cut on cryostat.

• Sections were incubated at 37оC for 45 minutes to 4 hours in the incubating

medium.

• Sections were rinsed in distilled water.

• Mounted in glycerin jelly.

Histochemical method for BChE

 Direct colouring method described by Karnovsky and Roots (1964) was adapted
for the demonstration of BChE activity.

Incubation medium:

5 mg of substrate (i.e. butyrylthiocholine iodide) was dissolved in 6.5 ml of 0.1M

phosphate buffer (pH = 6.0). Then following items were added in order while
stirring-

(1) Tri sodium citrate 0.1M = 0.5 ml

(2) Copper sulfate 0.03M = 1 ml

(3) Distilled water = 1 ml

(4) Potassium ferricyanide 0.005M = 1 ml

Procedure:

• 15 μm thick sections were cut on cryostat.

• Sections were incubated at 37оC for 45 minutes to 4 hours in the incubating

medium.

• Sections were rinsed in distilled water.

• Mounted in glycerin jelly.

Histochemical method for ATPase

The method of Wachstein and Meisel (1957) was used for demonstration of
ATPase.

Incubation medium:

ATP 0.125g % = 20 ml
Tris-maleate buffer (pH 7.2) 0.2M = 20 ml
Lead nitrate 2% = 3 ml
 Magnesium sulfate 0.1M = 5 ml
Distilled water = 2 ml

Procedure:

• 15 μm thick sections were cut on cryostat.

• Sections were incubated at 37оC for 5-60 minutes in the incubating medium.

• Sections were rinsed in distilled water.

• Then the sections were treated with 1% yellow ammonium sulphide for one
minute.

• Again the sections were rinsed in distilled water.

• Finally sections were mounted in glycerin jelly.