Turnover 10 times lower than the model general esterase substrate CMNA. Its strong chemical stability allows to quantify low picogram levels of hCE1 when using an endpoint measurement over 24 hours. Its turnover by hCE1 is at least 10-fold more selective compared to 13 different serine hydrolases tested and 40- to 50-fold more selective compared to hCE2
Title: Development of amide-based fluorescent probes for selective measurement of carboxylesterase 1 activity in tissue extracts Kodani SD, Barthelemy M, Kamita SG, Hammock B, Morisseau C Ref: Analytical Biochemistry, 539:81, 2017 : PubMed
Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.
