New fluorogenic substrate for esterase activity of α-chymotrypsin and related enzymes

✍ Scribed by Kenji Kuromizu; Yukiko Shimokawa; Okitoshi Abe; Nobuo Izumiya

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 436 KB
• Volume: 151
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90216-7

✦ Synopsis

A new fluorogenic substrate, benzyloxycarbonyl+phenylalanine 4-methylcoumaryl-7-ester, has been developed for determination of the esterase activity of ol-chymotrypsin and related enzymes. Synthesis of the substrate was achieved simply by the carbodiimide condensation of benzyloxycarbonyl+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 IOO-fold range of the enzyme concentration. As little as 2 ng of ol-chymotrypsin could be detected in the standard assay. A typical enzyme assay, stability of the substrate, kinetic parameters, and specific activity have been reported. 0 1985 Academic press. 1nc.