Purification and Partial Characterization of Human Pancreatic Elastase

Abstract

An elastolytic enzyme [EC 3.4.21.11] was isolated from human pancreatic juice. The concentration of the enzyme was .apprx. 0.6 mg/ml corresponding to .apprx. 10% of the protein concentration of the juice. The purification procedure included chromatography on Sephadex G-25 followed by ion-exchange chromatography on SP-Sephadex C-50 at pH 6.0, affinity chromatography on Trasylol-Sepharose 4-B and a final ion-exchange chromatography on SP-Sephadex at pH 7.6. The yield was .apprx. 50%. The elastase isolated was homogeneous in analytical disc electrophoresis and showed 1 protein component with a MW of 26,300 in dodecylsulfate-electrophoresis. There are similarities in the amino acid compositions of human and porcine pancreatic elastases. The human enzyme has a lower activity on elastin than porcine elastase but similar activities on casein and fibrin. The Km value for Boc-Ala-ONp was 5.13 .times. 10-4 M. The elastase isolated was 98% active, as judged from active site titration. The results of immunodiffusion studies of activated pancreatic juice and duodenal juice with specific elastase-directed antibodies indicate that the purified enzyme was the single elastolytic enzyme present. It is a cationic protein without any carbohydrate. The activation of the pro-elastase resulted in the formation of an active enzyme with a higher isoelectric point than the zymogen.