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. Carbonic Anhydrase?VB encoded by the CA5B gene is a mitochondrial protein. In comparison with another mitochondrial CA (CA5A), CA5B has different tissue distribution and chromosomal location. The activity of recombinant human CA5B 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 CA5B (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.