New fluorogenic substrate for esterase activity of alpha-chymotrypsin and related enzymes.
A new fluorogenic substrate, benzyloxycarbonyl-L-phenylalanine 4-methylcoumaryl-7-ester, has been developed for determination of the esterase activity of alpha-chymotrypsin and related enzymes. Synthesis of the substrate was achieved simply by the carbodiimide condensation of benzyloxycarbonyl-L-phenylalanine and 7-hydroxy-4-methylcoumarin in a 86% yield. The esterase activity was measured by increase of the fluorescence intensity at excitation and emission wavelengths of 325 and 465 nm, respectively. An initial rate of hydrolysis was linear over a 100-fold range of the enzyme concentration. As little as 2 ng of alpha-chymotrypsin could be detected in the standard assay. A typical enzyme assay, stability of the substrate, kinetic parameters, and specific activity have been reported.
D002918:Chymotrypsin / Q000378:metabolism*; D003374:Coumarins / Q000138:chemical synthesis*; D004950:Esterases / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D010196:Pancreatic Elastase / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013381:Subtilisins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D003374:Coumarins; D007202:Indicators and Reagents; D006923:Hymecromone; C047960:benzyloxycarbonylphenylalanine 4-methylcoumaryl-7-ester; D004950:Esterases; D013381:Subtilisins; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin