96-well plate assays for measuring collagenase activity using H-3-acetylated collagen

We describe two alternative assays for measuring collagenolytic activity us ing H-3-acetylated collagen. Both assays have been developed for the 96-wel l plate format and measure the amount of radiolabeled collagen fragments re leased into the supernatant from an insoluble 3H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments fro m the intact fibril by sedimentation of the undigested collagen by centrifu gation. The second method achieves this separation by filtration of the sup ernatant through the membrane of a 96-well filtration plate which retains t he undigested collagen fibril. Both methods give linear dose- and time-depe ndent responses of collagenase activity greater than or equal to 70% of tot al collagen lysis. In addition, both assays can be simply modified to measu re tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, whi ch is also linear between 20 and 75% of total collagen lysis with the amoun t of TIMP added. (C) 1999 Academic Press.