Immobilized fluorogenic substrates for proteinase: A method for visualization and quantitation of elastase release from human monocytes

An elastase-specific fluorogenic substrate, 6-(N-carbo-benzoxy-L-alanyl-L-alanyl-L-alanylamido)-qu inoline, was synthesized and immobilized via the fluorophoric group to an alkylatable derivative of polyacrylamide microspheres. Upon hydrolysis by elastase, the proteolytic product of the reaction fluorescences with a characteristic greenish-yellow light corresponding to the presence of the 1-alkyl-6-aminoquinolinium ion. This method has been applied to detect the elastase activity released from monocytes grown on the microspheres. Because the fluorescent product is covalently attached to the microsphere and cannot diffuse away from the site of reaction, it is possible to identify individual cells releasing the proteinase molecules. These experiments demonstrate that covalently immobilized fluorogenic substrates can be used for direct visualization and quantitation of proteinase activity from individual cells in culture.