Purification and preliminary characterization of human leukocyte elastasel.

Affinity chromatography permits the purification of 1–3 mg of human leukocyte elastase from the leukocytes contained in 500 ml of whole blood. Lysosomal granule proteins are extracted from polymorphonuclear leukocytes and subjected to chromatography on a column of elastin-Sepharose. Contaminating proteins are eluted with buffer containing 1 m NaCl and then elastase activity is eluted with buffer containing 8 m urea. The enzyme retains all of its esterase activity against N-t-BOC-l-alanine p-nitrophenyl ester after exposure to 8 m urea and retains 22% of its activity in the presence of 1% sodium dodecyl sulfate. In sodium dodecyl sulfate and 2-mercaptoethanol leukocyte elastase undergoes autolysis giving rise to several low molecular weight fragments. The molecular weight of the native enzyme is found to be 22.000 by both gel filtration and sodium dodecyl sulfate—acrylamide gel electrophoresis. A characteristic set of four isozymes is seen after acrylamide disc gel electrophoresis at pH 4.5. All bands are active against elastin and also contain carbohydrate by the periodic acid-Schiff stain. On the basis of stain intensity, the slower moving isozymes appear to be richest in carbohydrate. Active leukocyte elastase forms a complex with α₁-antitrypsin in a 1:1 molar ratio. The elastase must be enzymatically active for complex formation to occur.