AFFINITY CHROMATOGRAPHY IN THE SEPARATION OF HUMAN ALPHA1-ANTITRYPSIN (ALPHA-1-AT) AND ANTITHROMBIN-III (AT-III)

The two antiproteases α1-antitrypsin (α1-AT) and antithrombin-III (AT-III) have been purified simultaneously from human plasma. Purification procedure consisted of gel filtration on Sephadex G-200 after initial processing of plasma, followed by ion exchange chromatography on DEAE-Sephadex A50 and DEAE-cellulose, at a pH of 9.0 and pH 8.3 respectively. The two proteins could not be separated by any of these procedures including a lower pH (7.4) in ion exchange chromatography. Affinity chromatography on heparin-Sepharose separated the proteins since α1-AT did not bind to the matrix. Alpha1-AT unbound to the heparin-Sepharose was subsequently purified through con A-Sepharose affinity column. The final yield of both the proteins was about 20%. The molecular weight estimated on SDS electrophoresis for AT-III and α1-AT was 63,000 and 50,000, respectively.