Colorimetric and fluorimetric microplate assays for legumain and a staining reaction for detection of the enzyme after electrophoresis.

The cysteine endopeptidase legumain was recently discovered in mammalian cells, predominantly localized in the lysosomal system where it is believed to contribute to antigen processing for MHC class II. Here we describe rapid assay procedures for the enzyme in 96-well microplates with two substrates, a novel compound, succinyl-Ala-Ala-Asn-4-methoxy-2-naphthylamide, and benzyloxycarbonyl-Ala-Ala-Asn-4-methyl-7-coumarylamide. Both substrates are suitable for fluorimetric assays, but the naphthylamide also allows colorimetric detection of legumain activity, since the released 4-methoxy-2-naphthylamine gives a red product when coupled with the Fast Garnet color reagent. We show that the naphthylamide substrate can be used to visualize active legumain after electrophoresis in polyacrylamide gel. Both substrates provide assays that are reproducible and sufficiently sensitive to allow the assay of legumain in crude samples such as tissue homogenates, although the coumarylamide is the more sensitive. The specificity of both assay methods for legumain was verified by the lack of inhibition by E-64 and total inhibition by egg white cystatin.