PROTEINASE 3, NO. ML734
Also called AGP7, P29B and Myeloblastin (3)
Lot No. 505 Volume Lyophilized = 0.5 ml Weight = 1.0 mg PR3
ANALYTICAL DATA
60 Units per mg of protein.
Protein determined by coomassie blue method using as standard salt-free, detergent-free PR3.
EC 3.4.21.76
SOURCE. Leucocytes of Purulent Human Sputum
DESCRIPTION - LYOPHILIZED.
Highly purified by chromatography. Free of elastase and cathepsin G. Lyophilized from a solution containing 1 mg/ml of PR3 in 1mM Citric acid pH 3.5 + 0.005% Tritonâ X 100 + 0.25 M NaCl. The lyophilized product contains approximately 5% PR3 protein and 95% salts. Store lyophilized PR3 at -20°C, stable 12 months. May be freeze thawed 5 times.
Re-dissolve to 1mg/ml with 0.05 M citric acid pH 3.5 to pH 5.0 or H2O. Maintain solutions at 5 °C.
SDS-PAGE indicates MW = 29,000.
50 - 55 units per mg of protein. One unit will hydrolyze 1mMole of Boc-Ala-Ala-Nva-SBzl per minute at pH 7.5 at 25°C. The assay buffer-substrate (2 ml) is 0.1 M MOPS pH 7.5 containing 0.5 M NaCl, 10% DMSO, 0.25 mM substrate and 0.1 mM 5,5’-dithiobis-(2-nitrobenzoic acid). Use 0.2 to 0.5 mgm of enzyme. Read increase in absorbance at 405 nm. The 1.0 mM extinction coefficient is 13.1 in a 1.0 cm cell. The kinetics of PR3 hydrolysis of thiolbenzyl substrates is described by Kam et. al. (1).
Purity > 95% by native electrophoresis at pH 4.3 (4) yielding predominately 3 isoenzyme bands that migrate one-half the distance in comparison to Cathepsin G. Elastase, Lysozyme and Myeloperoxidase are also compared. A similar electrophoretic pattern was first described by Baggiolini et.al. (2) for the three proteases.
Enzyme Comparative Migration
Cathepsin G 1.0
Myeloperoxidase .34
Proteinase 3 .52
Elastase .69
Lysozyme .90
Amino terminal sequence ( IVGGHEAQPHSRPY ) confirms the identity of PR3.
NOTE: 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.