Preparing and optimizing a newly affinity gel for purification of carbonic anhydrase isoenzymes

Abstract

Human carbonic anhydrase isozymes have been purified from the hemolysate, directly by using the original affinity gel in chemical structure of Sepharose 4B-L-tyrosine- sulfathiazole. Different solution buffers were used for obtaining the purified CA isozymes from the affinity column. Most suitable elution buffers were determined for CA I and CA II isoenzymes pH 6.3, 25 mM Na2HPO4/1.0 M NaCl and pH 5.6, 0.1 M NaCH3COO / 0.5 M NaClO4 respectively. The purification values for CA I and CA II have been obtained as 635.71 and 666.71 folds with 28.41% and 46.19% yield respectively. The ionic strength and optimum pH values of the original affinity gel have been determined for each isozyme of CA. Maximum binding was achieved 0.3 ionic strength and pH 8.7 for both CA I and CA II. The single bands was indicated for each isoenzymes by SDS-polyacrylamide gel electrophoresis.