Carbonic Anhydrase

Carbonic anhydrases catalyze the reaction:

They are widespread in nature, being found in animals, plants, and certain bacteria. In animals they play an important role in respiration by facilitating transport of CO₂ and are involved in the transfer and accumulation of H⁺ and HCO^3-. In chloroplasts of plant cells their role may be related to photosynthetic fixation of CO₂. Lindskog et al. (1971) reviewed the carbonic anhydrases extensively.

Mammalian carbonic anhydrases are of several forms differing in enzymatic properties, amino acid sequences and inhibitor binding. See Nyman and Lindskog (1964) and Edwards (1990). In erythrocytes there is usually present both a high activity and a low activity form. The pH-rate profiles and pH-binding curves indicate that the same group with pKa of approximately seven is involved in both forms (Cohen et al. 1972). All the isoenzymes have a molecular weight of approximately 30,000 and contain one zinc atom per molecule. Although Deutsch et al. (1972) report six carbonic anhydrases in horse red blood cells, most interest has been focused on high and low activity types. Bovine erythrocytes contain two electrophoretically separable forms designated A and B in order of mobility. Both have a high order of activity similar to the human variant "C". Lindskog et al. (1971) devote a section of their review to polymorphism and nomenclature (pg. 595).

The outstanding characteristic of carbonic anhydrase is its very high turnover number (Khalifah 1973; Lindskog and Coleman 1973). Zinc may be replaced by cobalt, Whitney et al. (1967). Shinar and Navon (1974) found that the cobalt-substituted enzyme is dependent upon the state of oxidation of the metal. Appleton and Sarkar (1974) indicate the activity is related to the ionization of a group close to the zinc.

Characteristics of Carbonic Anhydrase from Bovine Erythrocytes:

Molecular weight: 30,000 (Lindskog et al. 1971).

Extinction coefficient: = 19.0 (Nyman and Lindskog 1964).

Inhibitors: Carbonic anhydrases are inhibited by most monovalent anions (Lindskog et al. 1971; Ward and Cull 1972) and sulfonamides (Binford et al. 1974). See also Pocker and Watamori (1973) on acetazolamide inhibition.

Specificity: Pocker et al. (1974) report that bovine carbonic anhydrase reversibly hydrates alkyl pyruvates and Wells et al. (1975) indicate that it exhibits hydratase activity toward a wide variety of substrates.

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

References