Purification and characterization of carbonic anhydrase from bovine erythrocyte plasma membrane

Carbonic anhydrase (CA) was purified from bovine erythrocyte plasma membran e and characterized in this study. For this purpose, the blood taken from y oung:animals was hemolysed, the membrane fraction was separated, and this f raction was repeatedly washed. The enzyme (CA) was removed from the membran e with buffered TritonX-100 (1%); it could be purified at a factor of 22.8 by affinity chromatography. The CA obtained from erythrocyte membrane has an esterase activity as well as hydratase activity. The V-max and K-m of the enzyme for the substrate (p -nitrophenyl acetate) are 1.948*10(-3) mM/ L*dak. and 3.596 mM, respectivel y. The purification degree of the enzyme was controlled by SDS-PAGE (3-10), which showed two distinct bands. It was determined that the enzyme had act ivity within the pH range of 4.5-9.5 and that the optimal pH was 7.5. The t emperature at which it showed activity was 20-60 degrees C and optimal temp erature was 37 degrees C. Molecular weight of CA was found to be 29844 and 61706 Dalton by gel filtration. On the other hand, sulfanilamide and acetaz olamide affected the enzyme.