HUMAN SPUTUM ELASTASE
NO. SE563
Lot No. 297, 379, 343 and lot 14994
EC 3.4.21.37
SOURCE. Leucocytes of Purulent Human Sputum.
SPECIFICATIONS.
Chromatographically purified. Lyophilized, salt-free, soluble powder. Store 5°C. Stable one year. Purity greater than 95% by SDS-PAGE. Molecular weight ca. 29,500. Free of Cathepsin G, MPO and lysozyme. 800 - 900 units per mg of protein on the substrate Suc-Al-Al-Al-pNA. Also, 15,000-19,000 units per mg of protein on the substrate MeO-Suc-Al-Al-Pro-Val-pNA. One unit will hydrolyze 1 Nmole of substrate per minute at pH 7.5 and 25°C.
PREPARATION.
Purulent human sputum is fractionated with NaCl and treated with DNast 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-Al-Al-Pro-Phe-pNA. The elastase from purulent human sputum was reported to possess similar enzymatic, inhibitory and physical chemical properities as elastase from human leucocytes (32).
DILUENTS AND STABILITY
1.) 1 mg per ml in 0.05 M NaOAc pH 5 containing 0.1 M NaCl. Stable at least 7
days at 5°C (in ice).
2.) 10 mg per ml in 50% glycerol 50% 0.02 M NaOAc pH5. Stable at least 60 days
at -20°C with 20 cycles between -20°C and 5°C.
3.) May be dissolved at 10 mg/ml in 2 mM HoAc and re-lyophilized in smaller
quantities.
Stability is better in acidic (»pH5) than basic buffers. Inactivation may occur at pH 8 due to autolysis and/or proteolysis by trypsin such as bovine pancreatic trypsin. Proteolysis can be inhibited by benzamidine. Autolysis and proteolysis can be inhibited by elastatinal. However, elastatinal is also an inhibitor of elastase (63). Low pH (»pH 5) protects the elastase from both autolysis and proteolysis.
REFERENCES.
(33) Anderson, K.K., Balny, C., Douzou,P., and Bieth, J., J.Chrom., 192, 236, (1980).
(62) Viscarello, R.L., Stein, R.L., Kusnel, E.LJ., Holsclaw, D., and Krell, R.D., Prep
Biochem. 13, 57, (1983).
(63) Stein, R.L., and Williams, J.C., Experientice 41, 638, (1985)
All of the human sputum leucocyte enzymes are prepared from human sputum shown to be non-reactive for HIV antibody, negative for Hepatitis C antibody.