Carbonic anhydrase (CA) catalyzes the reversible hydration and dehydration reactions of CO2/H2CO3. CAs are widespread in nature, being found in animals, plants, and certain bacteria. Sixteen isozymes have been identified and characterized in mammals. Since erythrocyte CA is relatively easy to obtain for experimental purposes, it is the most widely studied. Specificity Blood CO2 transport and excretion is largely dependent on the rapid catalysis of the CO2 reactions within the erythrocyte by CA. Bovine CA reversibly hydrates alkyl pyruvates and it exhibits hydratase activity toward a wide variety of substrates. Composition Sixteen CA isozymes have been described so far in mammals. Erythrocyte CAs, CA-I and CA-II, are most well known. CA-I, CA-II, CA-III, CA-VII, and CA-XIII are cytosolic. CA-IV, CA-IX, CA-XII, CA-XIV, and CA-XV are membrane bound. CA-VI is secreted in saliva. CA-VA and CA-VB are mitochondrial. There are also three acatalytic forms referred to as CA-related proteins (CARPs): CARP-VIII, CARP-X, and CARP-XI. The zinc metal is always bound to histidines 93, 95, and 118 (mature chain numbering). A hydrogen bonded network, linked to the zinc-bonded water molecule and these histidines either directly or indirectly, includes 28-Ser, 91-Glu, 105-Glu, 106-His, 116-His, 193-Tyr, 198-Thr, 208-Trp, and 223-Asn. These residues have been found to be highly conserved. Bovine and human CA I and II contain a unique C-terminal knot structure, which has been shown to be important in enzymatic and mechanical properties. Molecular Characteristics Each isozyme from erythrocytes (CA-I and CA-II) is composed of a single chain peptide of 259 or 260 amino acid residues. The low activity form (CA-I) contains 260 residues, while the high activity form (CA-II) contains 259 residues. Erythrocyte high and low activity forms within a given species usually show greater than 50% sequence identity; for example, equine CA-I and CA-II forms only show 55% identity. In contrast, the same forms from different species show much greater homology; human CA-II and bovine CA-II show 77% sequence homology. The isozymes are encoded by separate genes but given the great deal of homology, especially in the active center, they appear to have a common evolutionary history. Ubiquitin, a 76 residue protein with some enzymatic properties of CAs, has a distinct sequence homology to CA. Applications CO2 determination in blood Elimination of CO2 in reagents for acidity testing Carboxy group transfers Reduction reactions 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
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