A fluorescent screening assay for collagenase using collagen labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone.

R L O'Grady, A Nethery, N Hunter

Index: Anal. Biochem. 140(2) , 490-4, (1984)

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Abstract

This report describes the use of the compound 2-methoxy-2,4-diphenyl-3(2H)-furanone to label collagen as a substrate for the detection of mammalian collagenase in a fluorescent assay which is suitable for screening large numbers of samples. The compound 2-methoxy-2,4-diphenyl-3(2H)-furanone presents distinct advantages over other fluorophores, since both the unbound reagent and its hydrolysis products are nonfluorescent. The labeling procedure uses commercially available collagen, is fast and simple, and gives a 90% yield of labeled substrate. The fluorescent collagen substrate is stable and retains fluorescence over a wide range of pH. The assay detects, reproducibly, metal-dependent collagenase activity in microliter volumes of conditioned media from cultured neoplastic cells or in chromatographic fractions from such media.