Carbonic anhydrase (carbonate dehydratase) catalyzes the following reaction:

            CA
CO2 + H2O ------> H2CO3 

Mammalian erythrocytes contain two distinct forms of carbonic anhydrase distinguished by differences in their catalytic activities. The enzyme requires zinc for its activity and it has a molecular weight of 30,000.

1. 0.05 M Tris-HCl buffer, pH 7.6.
2. 0.003 M p-Nitrophenyl acetate. Dissolve 13.6 mg p-nitrophenyl acetate in 1 ml acetone and dilute with water to a volume of 25 ml. Prepare fresh daily and store in dark.
3. Carbonic anhydrase (enzyme) solution. Prepare in Tris buffer to yield a final concentration of 1-2 U/ml. Prepare fresh prior to assay.

1. Set spectrophotometer (equipped with a strip chart recorder and temperature control) at 348 nm and 25°C.
2. In a cuvette, pipette the following reagents:
   Tris-HCl buffer 1.9 ml
   p-nitrophenyl acetate 1.0 ml
   Incubate in spectrophotometer at 25°C for 5 min.
3. Record blank rate at 348 nm, if any.
4. Initiate the reaction by adding 0.1 ml enzyme solution (1-2 U/ml) to the cuvette. Record the absorbance at 348 nm for 5 min.
5. Calculate E348 nm/min