Cholinesterase, Acetyl Assay

Method: The rate of acid production resulting from the hydrolysis of acetylcholine is measured titrimetrically. One unit hydrolyzes one micromole of acetylcholine per minute at 25deg.C and pH 7.0 under the specified conditions.

Reagents

• 0.02 M Potassium phosphate, pH 7.0
• 0.005 M Acetylcholine
• 0.01% Gelatin, containing 0.2 M sodium chloride and 0.04 M magnesium chloride
• 0.01-0.02 N Sodium hydroxide, standardized

Enzyme

Dissolve at 1 mg/ml in 0.02 M potassium phosphate, pH 7.0. Dilutions are made in 0.02 M potassium phosphate buffer, pH 7.0 to obtain a response between 0.05-0.2 ml/min. immediately before use.

Procedure

The titration can be carried out with either an automatic titrator or a laboratory pH meter. The reaction vessel must be maintained at 25°C.

Pipette the following into a 25 ml beaker:

0.005 M Acetylcholine	7.5 ml
0.01% Gelatin-salt mixture	7.5 ml

At zero time, add 0.1-1.0 ml of appropriately diluted enzyme and adjust the pH to 7.0.

After a constant rate is achieved, record the volume of base required to maintain the pH at 7.00 for 5-6 minutes.

Calculation