A novel and simple assay for determination of gelatinase-b (mmp-9) activities in biological fluids
article
Here we describe a new principle to assess specifically the activity of the different members of the human matrix-metalloproteinase family (MMPs) by a colorimetric assay. Using protein engineering a modified pro-urokinase has been made in which the activation sequence, normally recognized by plasmin (PRFK IIGG) has been replaced by a sequence that will be recognized by MMPs (RPLG IIGG) The active urokinase resulting from the activation of modified pro-urokinase by MMPs can be measured directly using a specific chromogenic peptide substrate for urokinase. Next to overall collagenase activity the assay can be made specific for the various MMPs using monoclonal antibodies and/or specific sites of hydrolysis. Following catching by specific antibodies of a particular MMP from biological fluids or tissue culture media, MMP-activity of both active and 'to be activated' MMP can be analyzed. Due to the catching no side-effects of disturbing factors in the biological fluids will interfere with the assay. Using this assay a significant increased level of both active and latent MMP-9 could be measured in saliva from patients with Sjögren syndrome compared with normals (2.46 vs 0.8 p<0.05 relative units for active MMP-9 and 26.2 vs 9.4 p<0.0001 for active plus latent MMP-9). Total MMP-9 in breast tumor extracts was found to be considerably higher in malign tumors as compared with benign tumors (21.5 vs 6.3 relative units p=0.0002). Similarly, increased MMP-9 levels were found in stomach tumor tissue as compared with normal stomach tissue. This assay can be adapted for the specific detection of all the different members of the family of MMPs or other proteases.
TNO Identifier
234226
ISSN
13690191
Source
Fibrinolysis and Proteolysis, 11(SUPPL. 3), pp. 41.
Pages
41
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