SOURCE. Leucocytes of Purulent Human Sputum.
Also called AGP7, P29B and Myeloblastin (3).
Highly purified by chromatography. Free of elastase and cathepsin G. A solution of PR3 in 0.05 M NaOAc pH 5 containing 0.6 M NaCl. Protein determined by coomassie blue method using as standard bovine serum albumin. Store PR3 at -20°C, stable 12 months. MW = 29000.
Maintain solutions at 5°C.
SDS-PAGE indicates MW = 29000.
greater than 50 units per mg of protein.
One unit will hydrolyze 1 µMole of Boc-Ala-Ala-Nva-SBzl (EPC No. FA327) 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 µgm of enzyme. Read increase in absorbance at 405 nm. The 1.0 mM extinction coefficience is 13.1 in a 1.0 cm cell. The kinetics of PR3 hydrolysis of thiobenzyl substrates is described by Kam et al. (1).
Purity >95% by native electrohoresis 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 and Lysozyme, .90.
ML734, Proteinase 3, Quantities bases on PR3 content.
(1) Kam C., Kerrigan, J.E., Dolman, K.M., Goldschmeding, R., Von dem Borne, Albert E.G.Kr., Powers,J.C., Vol 297, number 1,2, 119-123, February 1992.
(2) Baggiolini, M., Bretz, U., Dewald, B., and Feigenson, M.E., Agents and
Actions, Vol. 81/2 pg. 3-10,(1978)
(3) Bories, D., Raynal,, M.-C., Solomon, D.H., Darzynkiewizc, Z., & Cayre, Y.E.,. (1989) Cell 59, 959.
(4) Reisfeld, R.A., Lewis, U.J., and Williams, D.E., Nature, No. 4838, pg. 281-2.(1962).
Note. All human sputum leucocyte enzymes are prepared from human sputum shown to be non-reactive for HIV antibody, negative for Hepatitis C antibody.