The Evaluation of Inhibition Effects of Antibiotics on the Sheep Stomach Carbonic Anhydrase I and II Isozymes

Abstract

Carbonic anhydrases are vital class of enzymes that participate in so many essential physiological events in the organism and associated with many diseases. Inhibitors of carbonic anhydrase enzymes are used in pharmacological applications in many areas such as antiobesity, antiglaucoma, anticancer agents and anticonvulsants. So, this study focuses on the characterization of CA-I and CA-II isoenzymes purified from sheep stomach and investigating the inhibition effects of antibiotics on these enzymes. The findings show that the antibiotics studied strongly inhibit CA-I and CA-II isozymes. In our presented study, using Sepharose 4B-L-Tyrosine sulfanilamide affinity gel chromatography, carbonic anhydrase I and carbonic anhydrase II isoenzymes were purified from sheep stomach with a yield of 51.9%, 78%, respectively and specific activity of CA I and CA II are 4864.8 EU/mg and 5652.02 EU/mg, respectively. The overall purifications from sheep stomach CA I and CA II were approximately 529.4 and 615.2 fold respectively. To check the purify of the enzymes, it was conducted an electrophoretic method so-called Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and single bands were detected for both enzymes. Additionally, the effects of antibiotics on sheep stomach CA I and CA II isozymes activities, using both hydratase and esterase activity methods, were researched. IC50 values of enrofloxacin, tylosin and ampicillin antibiotics that inhibit CA I enzyme with hydratase activity were found as 1.41, 0.033 and 1.56 mM, respectively. IC50 values for CA II enzyme with hydratase activity of the same antibiotics were found as 2.47, 0.039 and 1.63 mM, respectively. Kanamycin and amikacin antibiotics inhibited both CA I and CA II enzymes with esterase activity. IC50 values for CA I and CA II enzymes with esterase activity of kanamycin antibiotic were determined as 0.0488 and 0.118 mM, respectively. IC50 values of amikacin for CA I and CA II enzymes with esterase activity were determined as 0.0163 and 0.036 mM, respectively.