High-performance affinity chromatography was performed on five ligand-bound columns in an attempt to purify tissue-type plasminogen activator (t-PA), which is a glycoprotein with a high affinity for fibrin and also has two Kringle structures and a figer-domain in its molecule. The five high-performance affinity chromatography columns prepared were: concanavalin A-5PW, p-aminobenzamidine-5PW, iminodiacetic acid-5PW, boric acid-5PW, and lysine-5PW. All of the five high-performance affinity chromatographies were able to separate t-PA from contaminating proteins rapidly with high resolution and recovery. It was found that the combination of iminodiacetic acid-5PW, concanavalin A-5PW and p-aminobenzamidine-5PW gave excellent purification. The purification factor was about 300, and the recovery rate was 27.5%. The purity of the final product was checked by SDS-PAGE and electrophoretic enzymography, which revealed a single band with a molecular weight of 72, 000. The above combination of affinity chromatographies is thus useful for the rapid and convenient purification of t-PA.
