QUANTITATIVE REVERSE ZYMOGRAPHY - ANALYSIS OF PICOGRAM AMOUNTS OF METALLOPROTEINASE INHIBITORS USING GELATINASE-A AND GELATINASE-B REVERSE ZYMOGRAMS

Matrix metalloproteinases are a growing family of neutral pH optima, z inc atom-dependent endopeptidases that collectively degrade all compon ents of the extracellular matrix. This family of related proteases is further defined by their inhibition of protease activity by a class of low-molecular-weight endogenous inhibitors known as tissue inhibitors of metalloproteinases or TIMPs, Reverse zymography is an electrophore tic technique used to identify TIMP inhibitory activity within acrylam ide gels. Previous methods have generally used biochemically complex s ources of proteolytic activity (such as cell culture conditioned media ) copolymerized with a proteinase substrate in the gel to identify the zones of inhibited proteolysis. We describe a novel system for revers e zymography using purified recombinant human gelatinase A or gelatina se B in place of conditioned media. These reverse zymograms using reco mbinant gelatinase have sensitivities for TIMPs that are favorable in comparison to immunoblotting techniques but have the benefit of visual izing multiple inhibitors simultaneously. We have developed and charac terized these methods for the evaluation of inhibitors and have shown them to be highly sensitive, convenient, and reproducible. Both system s detect TIMPs 1, 2, and 3 simultaneously, but with differential sensi tivities for TIMPs 1 and 2. Using gelatinase A the system can detect a s little as 1 pg of rTIMP-8, but the limit of detection for rTIMP-1 is 40 pg. Gelatinase B shows less differential activity in that the limi ts of detection are 60 and 40 pg for TIMP-2 and TIMP-1, respectively. We demonstrate how these varied sensitivities of the gelatinases for t he TIMPs can contribute to potential pitfalls in systems using unchara cterized reagents (i.e., conditioned media). (C) 1997 Academic Press, Inc.