ML734, Proteinase 3

PROTEINASE 3, NO. ML734

Also called AGP7, P29B and Myeloblastin (3)

Lot No.   508        Volume Lyophilized = 1.0  ml      Protein= .011 mg/ml

ANALYTICAL DATA

650 Units per mg of protein.

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  + 0.25 M NaCl.  The lyophilized product contains approximately 5% PR3 protein and 95% salts.  Protein determined by coomassie blue method using as standard bovine serum albumin.  Store lyophilized PR3  at  -20°C, stable 12 months. 

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.

>600 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

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.

 References:

1. Kam, C., Kerrigan, J.E., Dolman, K.M., Goldschmeding, R., Von dem Borne, Albert E.G.Kr., Powers, J.C., FEBS (Federation of European Biochemical Societies) Vol 297, number 1, 2, 119-123, February 1992.
2. Baggiolini, M., Bretz, U., Dewald, B., and Feigenson, M.E., Agents and Actions, Vol 8½, pg. 3-10, (1978).
3. Bories, D., Raynald, M.C., Solomon, D.H., Darzynkiewizc, Z., and Cayre, Y.E., (1989) Cell 59, 959.
4. Reisfeld, R.A., Lewis, U.J., and Williams, D.E., Nature No. 4838, pg. 281-283, (1962).