Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K_m and V_max values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K_m values at optimum pH and 20°C were 0.625?mM, 0.541?mM, 0.785?mM and 0.862?mM with p-nitro phenyl acetate, for all CA localizations. The respective V_max values at optimum pH and 20°C were 0.875?μmol/L?min, 0.186?μmol/L?min, 0.214?μmol/L?min and 0.253?μmol/L?min with the same substrate. The K_i and I₅₀ values for the inhibitors sulphanilamide, KSCN, NaN₃ and acetazolamide were determined for all the CA localizations.