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Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.

Bioorganic & medicinal chemistry (2012-09-15)
Ramazan Demirdağ, Veysel Çomaklı, Murat Şentürk, Deniz Ekinci, Ö İrfan Küfrevioğlu, Claudiu T Supuran
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Sulfanilamide, VETRANAL®, analytical standard
Sigma-Aldrich
Sulfanilamide, puriss. p.a., ≥99% (calc. to the dried substance)
Sigma-Aldrich
Sulfanilamide, ≥98%
Supelco
Sulfanilamide melting point standard, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Sulfanilamide, Vetec, reagent grade, 97%