Matrix metalloproteinases are active following guanidine hydrochloride extraction of cartilage: generation of DIPEN neoepitope during dialysis

We have recently observed marked increases in MMP-derived aggrecan fragments in extracts of cartilage stimulated with IL-1. The fragments were detected with an anti-DIPEN neoepitope antibody that is specific for fragments generated by MMP cleavage at the DIPEN341 down arrow F(342)FGVG site. Because our results contrasted with another study, we systematically compared our methods with other published methods. We now report that DIPEN341, neoepitope can be generated post-culture, by dialysing GuHCl(1)-denatured samples against unbuffered, deionised water at 4 degreesC. We show that EDTA must be included in the GuHCl extractant, as well as the dialysis buffer, in order to block post-culture processing of aggrecan by MMPs. (C) 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved.