Purification and Characterization of Carbonic Anhydrase from A. gri Balik Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Abstract

Carbonic anhydrase (CA) was purified from Ar Balk Lake trout gill (fCA) by affinity chromatography on a sepharose 4B-tyrosine-sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg-protein)(-1), and a yield of 79.3 by using sepharose-4B-l tyrosine-sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was found to be about 29.9 kDa. The kinetic parameters, K-M and V-max were determined for the 4-nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The K-i constants for mafenide (1), p-toluenesulfonamide (2), 2-bromo-benzene sulfonamide (3), 4-chlorobenzene sulfonamide (4), 4-amino-6-chloro-1-3 benzenedisulfonamide (5), sulfamethazine (6), sulfaguanidine (7), sulfadiazine (8), and acetozazolamide (9) were in the range of 7.5-108.75 M. (C) 2014 Wiley Periodicals, Inc.