Demir, Nazan; Demir, Yasar; Coskun, Fatma; Tasgin, Esen

Carbonic anhydrase is an enzyme that takes responsibility in inhalation function but, until today, carbonic anhydrase is not examined if it is present in the bovine brain membrane or not. The enzyme carbonic anhydrase was purified and separately characterized according to the bonding forms in 4 steps such as outer peripheral, cytosolic, inner peripheral and integral. Affinity chromatography was used for purification of the enzyme in all four steps. The affinity column was prepared with sepharose-4B-L-tyrosine-sulphanilamide. Purified carbonic anhydrase was obtained at each step. Enzyme activity was measured by CO2 hydratase activity and esterase activity methods. Optimum pH and optimum temperature were defined for purified enzymes at each step. Morover molecular weight and purity were detected by gel filtration and SDS-PAGE electrophorose. In addition, the enzyme's Km and nmax values were found with the Lineweaver- Burk method. The obtained results are discussed in comparison with other mammalian carbonic anhydrases. Carbonic anhydrase was shown to be exist in bovine brain membrane and this enzyme was optimized.

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Tasgin, Esen; Nadaroglu, Hayrunnisa; Demir, Yasar; Demir, Nazan

In this work, the carbonic anhydrase was purified from bovine bone marrow and investigated its kinetic properties. Carbonic anhydrase was purified from bovine bone marrow using affinity chromatography by sepharose 4B-L-tyrosine sulphanilamide. During purification steps, the activity of enzyme was measured using p-nitrophenyl acetate at pH: 7.4. Optimum pH and optimum temperature values for bovine bone marrow carbonic anhydrase were determined and then K m and Vmax values for the same substrate were obtained by means of Linewearver-Burk graphics. The purification degree for bovine bone marrow was calculated. The Vmax, and Km values at optimum pH and at 20 °C for the substrate (p-nitrophenyl acetate) were 120.418 uμmol/L min and 2.409 x 103 mM, respectively. The K1 and I50 values for sulfanilamide, KSCN, NaN3 and acetazolamide were determined in bovine bone marrow carbonic anhydrase.

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