Cholinesterase, Butyryl Assay

A number of assays have been developed for BChE, including Grubic et al. 1975, Augustinsson and Ericksson 1974, Smith 1974, Brownson and Watts 1973, Dietz et al. 1973, Ellin 1972, Baum and Ward 1971, Garry 1971, Garry et al. 1971, and Hanss and Rey 1971 who report on a conductivity method. The following assay is used at Worthington. 

Method: The reaction velocity is determined by a titrimetric measurement of the acid released during the hydrolysis of acetylcholine. One Unit causes hydrolysis of one micromole of acetylcholine per minute at 25°C and pH 7.4, under the specified conditions.

Reagents

• 0.02 M Tris⋅HCl buffer, pH 7.4
• 0.2 M Magnesium chloride
• 2.2 M Acetylcholine chloride
• Titrant: 0.01-0.02 N NaOH, standardized

Enzyme

Dissolve at one mg/ml in reagent grade water.

Procedure

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

Pipette the following into a titration vessel at 25°C

Reagent grade water	7.0 ml
0.2 M Magnesium chloride	3.0 ml
0.02 M Tris⋅HCl buffer, pH 7.4	3.0 ml
Enzyme	0.1-0.5 ml

Adjust pH to 7.5 with titrant. At zero time add 1 ml of 2.2 M acetylcholine and adjust pH to 7.4. Record the amount of standard base required to maintain the pH at 7.4 for 5-6 minutes after a constant rate is achieved.

Calculation