SOURCE. Leucocytes of Purulent Human Sputum. EPC No. SE563 covalently labeled with fluorescein-isothiocyanate (Isomer 1). Non-bound FITC is removed by size exclusion chromatography.
Chromatographically purified. Lyophilized, salt-free,
Store at 5°C. Stable one year.
Purity greater than 95% by SDS-PAGE.MW ca. 29,500.
Free of cathepsin G, MPO and lysozyme.
1). Using Suc-Ala-Ala-Ala-pNA (EPC No. NS945); 800-900 units per mg of protein; one unit will hydrolyze 1 nM of substrate per minute at pH 7.5 and 25°C.
2). Using MeO-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237); 16-18 units per mg of protein; one unit will hydrolyze 1 µM of substrate per minute at pH 7.5 and 25°C. Assay procedures are given in Methods.
Purulent human sputum is fractionated with NaCl and treated with DNase to release DNA-bound proteins. The elastase is purified by affinity chromatography on Agarose-DAPSA-Elastin Soluble (EPC Product No. AD477) and separated from cathepsins by ion exchange chromatography on carboxymethyl-agarose (62). The elastase is further purified by affinity chromatography at -15°C on agarose-alanyl-alanyl-alanine (33). The elastase is free of cathepsin G activity when assayed with Suc-Ala-Ala-Pro-Phe-pNA. The elastase from purulent human sputum was reported to possess similar enzymatic, inhibitory and physical chemical properties as elastase from human leucocytes (32).
DILUENTS AND STABILITY.
- 1.) 1mg/ml in 0.05 M NaOAc pH 5 containing 0.1 M NaCl. Stable 7 days at 5°C (in ice).
- 2.) 10mg/ml in 50% glycerol 50% 0.02 M NaOAc pH 5. Stable at least 60 days at -20°C with 20 cycles between -20°C and 5°C.
- 3.) May be dissolved at 10mg/ml in 2 mM HoAc and re-lyophilized in smaller quantities.
Stability is better in acidic (~pH 5) than basic buffers. Inactivation may occur at pH 8.0 due to autolysis and/or proteolysis by trypsin such as bovine pancreatic trypsin. Proteolysis can be inhibited by benamidine. Autolysis and proteolysis can be inhibited by elastatinal. However, elastatinal is also an inhibitor of elastase (63). Low pH (~5) protects the elastase from both autolysis and proteolysis.