Detection of proteolytic activity by fluorescent zymogram in-gel assays

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluoresc ent zymogram in-gel protease assays, using SDS polyacrylamide gels copolyme rized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and mol ecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copol ymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buff er, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was defected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these prote ases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from se cretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substr ate. Overall, this sensitive fluorescent zymogram in-gel protease assay met hod can be used for rapid determination of protease cleavage specificity an d enzyme molecular weight in biological samples. This assay should be usefu l for many research disciplines investigating the role of the many protease s that control cellular functions.