Purification and characterization of carbonic anhydrase enzyme from goat liver and kidney tissues and investigation of the effects of some metal ions, antibiotics and avermectins on the enzyme activity

In this thesis, carbonic anhydrase (CA) enzyme was purified from goat liver and kidney tissues with a specific activity of 61229.29 and 58974.33 EU/mg and a yield of 55.6% and 51.0% by using Sepharose-4B-L-tyrosinesulfanilamide affinity chromatography, respectively. The overall purifications from goat liver and kidney tissues CA enzymes were approximately 359.18 and 212.47 fold purified respectively. For determination of enzyme purity and subunit molecular weights, SDS-PAGE was performed and single band was absorved for each tissue enzyme. The molecular weight of subunit of enzymes were evaluated as 27.70 kDa for liver and 28.00 kDa for kidney tissues. Optimum ionic strengths for CA enzymes from liver and kidney tissues were obtained as 0.5 M and 0.6 M Tris-SO4 buffer respectively. Optimum and stable pH values were determined as pH 7.5 and 7.0, respectively. The optimum temperature, activation enthalpy (∆H), activation energy (Ea) and Q10 values were obtained from Arrhenius plot for CA enzymes. Then, Km and Vmax values for p-nitrophenilacetate of the purified enzymes were calculated from Lineweaver-Burk graphs. Kcat values of the goat liver and kidney tissues CA enzymes were determined as 1328.51 and 906.66 s-1 respectively. Also, the inhibitory effects of some heavy metal ions (Pb2+, Co2+, Fe2+, Hg2+, Cu2+ and Ag+) antibiotics (amoxicillin and vancomycin) and avermectin (moxidectin, doramectin, abamectin and eprinomectin) on goat CA enzyme activities were evaluated by using esterase method. IC50 and Ki values of metals, antibiotic and avermectins, which showing the inhibition effect were calculated.