Carbonic Anhydrase Assay

Method: The electrometric method of Wilbur and Anderson (1948) in which the time required (in seconds) for a saturated CO₂ solution to lower the pH of 0.012 M Tris⋅HCl buffer from 8.3 to 6.3 at 0°C is determined. The time without enzyme is recorded at T₀; with enzyme, T.

Reagents

• 0.02 M Tris⋅HCl buffer, pH 8.0. Store in an ice bath at 0-4°C before and during use.
• Carbon dioxide saturated water. Bubble CO₂ gas through 200 ml ice cold water for 30 minutes prior to assay. During saturation process, store water at 0-4°C in an ice bath.

Enzyme

Dissolve lyophilized powder at a concentration of 0.1 mg/ml in ice cold water. Store in ice bath prior to use. IMMEDIATELY prior to use dilute suspensions or lyophilized materials to a concentration of approximately 0.01 mg/ml in ice cold water.

Procedure

Blank Determination: Add 6.0 ml of chilled 0.02 M Tris⋅HCl buffer, pH 8.0 to a 15-20 ml beaker. Maintain temperature at 0-4°C and record pH.

Withdraw in a 5 ml syringe, 4 ml of chilled CO₂ saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T₀.

Enzyme Determination: Add 6.0 ml of chilled 0.02 M Tris⋅HCl buffer, pH 8.0 to a 20 ml beaker. Maintain temperature at 0-4°C and record pH. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO₂ saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.

Calculation