chondroitin-sulfates and 1-10-phenanthroline

Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin 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 microM) and U24522 (IC50 = 9 microM) inhibited degradation, while phosphoramidon 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 substrates such as cartilage matrix.

Topics: Animals; Antibodies, Monoclonal; Cartilage, Articular; Chondroitin Sulfates; Enzyme-Linked Immunosorbent Assay; Femur; Glycopeptides; Humans; Hyaluronic Acid; Matrix Metalloproteinase 3; Metalloendopeptidases; Mice; Phenanthrolines; Proteoglycans; Recombinant Proteins; Substance P