A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD₄K-conjugated 7-amino-4-methylcoumarin

Abstract

Fig. 1. Structure of 2-naphthylamine (A) and 7-amino-4-methylcoumarin (B).A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD 4 K-conjugated 7-amino-4-methylcoumarin Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys (D4K). The assay for enteropeptidase has utilized GD4K-conjugated 2-naphthylamine (GD4K-NA) as a fluorogenic probe over the last 30 years. However, no other D4K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of GD4K-conjugated 7-amino-4-methylcoumarin (GD4K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a KM of 0.025 mM and a kcat of 65 sec -1 for GD4K-AMC, whereas it has a KM of 0.5 to 0.6 mM and a kcat of 25 sec -1 for GD4K-NA. The optimum pH of GD4K-AMC hydrolysis was pH 8.0. Our data indicate that GD4K-AMC is more suitable as a substrate for enteropeptidase than GD4K-NA. [BMB reports 2011; 44(7): 458-461]

Discovery and characterization of a small‐molecule enteropeptidase inhibitor, SCO‐792

“…All enteropeptidase enzyme assay and compound evaluation were conducted at pH 8 because the optimal pH of enteropeptidase was 8 as previously reported; Magee et al …”

The application of a UHPLC system to study the formation of various chemical species by compounds undergoing efficient self-aggregation and to determine the homodimerization constants (KDM) with values in the high range of 106–1010 M−1

“…C120 is also a well-known laboratory reagent, employed as a uorogenic probe for detection of enzymatic activity. 51 This dye has been used as a uorescence probe to analyze glycoproteins, monosaccharides and N-linked oligosaccharides via chromatography. 52 C120 has also shown effective antitubercular, 53 antibacterial and antifungal 54 activities.…”