Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75\%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS-PAGE was found to be 29kDa. The kinetic parameters, K(M) and V(max) values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The K(i) constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348-69.31μM.
Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity / R. Demirdağ;V. Comaklı;M. Sentürk;D. Ekinci;O. I. Küfrevioğlu;C. T. Supuran. - In: BIOORGANIC & MEDICINAL CHEMISTRY. - ISSN 0968-0896. - STAMPA. - 21:(2013), pp. 1522-1525. [10.1016/j.bmc.2012.08.018]
Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.
SUPURAN, CLAUDIU TRANDAFIR
2013
Abstract
Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75\%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS-PAGE was found to be 29kDa. The kinetic parameters, K(M) and V(max) values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The K(i) constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348-69.31μM.I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.