One unit of enzyme activity is defined as the 1 μg of enzyme required to convert 1 pmol of 4-Nitrophenyl acetate to 4-Nitrophenol in 1min at 37°C. The specific activity of recombinant rat CA4 is > 200 pmol/min/μg.

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. CA4 is a GPIanchored membrane enzyme expressed on the luminal surfaces of pulmonary (and certain other) capillaries and of proximal renal tubules. It functions as the principal CO2?taste sensor. The activity of recombinant rat CA4 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 CA4 (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.