A STROMELYSIN ASSAY FOR THE ASSESSMENT OF METALLOPROTEASE INHIBITORS ON HUMAN AGGREGATED PROTEOGLYCAN

Human proteoglycan was aggregated to an immobilized hyaluronan solid p hase on a 96-well ELISA plate. This complex was then degraded by recom binant human stromelysin. The remaining proteoglycan fragments were de tected using a monoclonal antibody probe directed against the chondroi tin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52 muM) and U24522 (IC50 = 9 muM) inhibited degradation, while phosphoram idon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrat es such as cartilage matrix.