title: Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.
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, KM and Vmax values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The Ki 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.
authors.affiliation: Agri Ibrahim Cecen University, Vocational Training School, Agri, Turkey. r.demirdag@hotmail.com
authors.forename: Ramazan
authors.initials: R
authors.lastname: Demirdağ
authors.forename: Veysel
authors.initials: V
authors.lastname: Çomaklı
authors.forename: Murat
authors.initials: M
authors.lastname: Şentürk
authors.forename: Deniz
authors.initials: D
authors.lastname: Ekinci
authors.forename: Ö
authors.initials: Ö
authors.lastname: İrfan Küfrevioğlu
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
mesh_terms: D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D012756:Sheep; D000077145:Sulfanilamide; D013424:Sulfanilamides / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D013424:Sulfanilamides; D000077145:Sulfanilamide; D002256:Carbonic Anhydrases