Carbonic Anhydrase (CA) catalyzes the reversible reaction of CO2? H2O = HCO3-? H , which is fundamental to many processes such as respiration, renal tubular acidification and bone resorption. CA2 is a cytosolic enzyme with the highest activity among all known CAs. Mutations in the CA2 gene result in the CA II deficiency syndrome, an autosomal recessive disorder that produces osteopetrosis, renal tubular acidosis and cerebral calcification. The activity of recombinant rat CA2 was measured by its ability to hydrolyze 4-Nitrophenyl acetate (4-NPA) to 4-Nitrophenol. The reaction was performed in 12.5 mM Tris, 75 mM NaCl, pH 7.5 (assay buffer), initiated by addition 50 μL of various concentrations of CA2 (diluted by assay buffer) to 50 μL of 2 mM substrate 4-NPA (100 mM stock in Acetone, diluted by assay buffer). Incubated at 37℃ for 5min, then read at a wavelength of 400 nm.