Purification and characterization of carbonic anhydrase from human erythrocyte plasma membrane

Carbonic anhydrase is the basic enzyme in inhalation function. Untill now no research had been done to determine whether CA is in the human erythrocyte plasma membrane are not. Carbonic anhydrase (CA) was purified from human erythrocyte plasma membrane and describe in this study. For this purpose, the blood samples taken from young human test subjects were hemolyzed, then the membrane fraction was separated, and this fraction was repeatedly washed. The enzyme (CA) was remove from the membrane with buffered TritonX-100 (1%), with was purified with a factor of 119.19 by affinity chromotography. The CA abtained from the erythrocyte membrane has esterasse activity as well as hydratase activity. The Vmax and KM enxyme for the subtrate (p-nitrophenyl acetate) are 1.517*10-1 μM/L*min and 1.78 mM, respectively. The purification degree of the enzyme was controlled by SDS-PAGE (3-10%), which showed one distinct band. It was determine that the enzyme was active within the pH range of 4-10, and that the optimal pH was 7.5. The temperature at which it showed activity was 5-70°C, and the optimal temperature was 35°C. The molecular weight of CA wasfound to be ∼ 36.600 by gel filtration. On the other hand, sulphanilamide, KSCN and NaN3 inhibited the enzyme. Finally, CA was shown to be present in human erythrocyte plasma membrane and this enzyme is optimized.