Purification of human neutrophil collagenase and production of a monospecific antiserum.
Although there is good evidence for the presence of human neutrophil (PMN) collagenase, only moderate purification has been reported. The probable explanation for this fact is that most assays used to specifically measure collagenase activity are not reliable if high levels of several different proteases are also present in the assay mixture. The PMN granule is just such a concentrated mixture. Therefore, polyacrylamide gel electrophoresis was used to identify and quantitate the alpha 1 3/4 and alpha 2 3/4 cleavage products diagnostic for mammalian collagenase. White cells (85% PMN's) were lysed in 0.34 M sucrose and granules were obtained. The granules were lysed by sonication, and the lysate was chromatographed on a Sephadex G-200 column followed by a Trasylol-Sepharose 4B column. This procedure resulted in a 1350-fold purification and a yield of 75 micrograms of enzyme/unit of blood. The collagenase was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid but not by sulfhydryl or serine protease inhibitors. The preparation was free of elastase, which has been shown to cleave type III collagen into alpha 1 3/4 and alpha 1 1/4 pieces. The pI of collagenase was shown to be 4.7 by isoelectric focusing, and the enzyme lost activity below a pH of 6.5 if collagen was absent. Antiserum was produced by 100-micrograms injections of the purified collagenase into rabbits. Titers were measured by the enzyme-linked immunosorbent assay. For determination of the specificity, collagenase and PMN extract were isoelectrically focused and blotted onto nitrocellulose. The antibody recognized only one band of protein in the PMN extract, which comigrated with the purified collagenase.
D000818:Animals; D000918:Antibody Specificity; D002845:Chromatography; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007526:Isoelectric Point; D003012:Microbial Collagenase / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000302:isolation & purification; D011817:Rabbits / Q000276:immunology
D007106:Immune Sera; D010196:Pancreatic Elastase; D003012:Microbial Collagenase