7-Amino-4-methylcoumarin
7-Amino-4-methylcoumarin is more suitable as a substrate for enteropeptidase than GD(4)K-NA.
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Providing storage is as stated on the product vial and the vial is kept tightly sealed, the product can be stored for up to
24 months(2-8C).
Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20C. Generally, these will be useable for up to two weeks. Before use, and prior to opening the vial we recommend that you allow your product to equilibrate to room temperature for at least 1 hour.
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The packaging of the product may have turned upside down during transportation, resulting in the natural compounds adhering to the neck or cap of the vial. take the vial out of its packaging and gently shake to let the compounds fall to the bottom of the vial. for liquid products, centrifuge at 200-500 RPM to gather the liquid at the bottom of the vial. try to avoid loss or contamination during handling.
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BMB Rep. 2011 Jul;44(7):458-61.
A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin.[Pubmed:
21777516]
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 (D(4)K). The assay for enteropeptidase has utilized GD(4)K-conjugated 2-naphthylamine (GD(4)K-NA) as a fluorogenic probe over the last 30 years. However, no other D(4)K-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans.
METHODS AND RESULTS:
In this study, we used shift in the emission spectrum of GD(4)K-conjugated 7-Amino-4-methylcoumarin (GD(4)K-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a K(M) of 0.025 mM and a k(cat) of 65 sec(-1) for 7-Amino-4-methylcoumarin, whereas it has a K(M) of 0.5 to 0.6 mM and a k(cat) of 25 sec(-1) for GD(4)K-NA. The optimum pH of 7-Amino-4-methylcoumarin hydrolysis was pH 8.0.
CONCLUSIONS:
Our data indicate that 7-Amino-4-methylcoumarin is more suitable as a substrate for enteropeptidase than GD(4)K-NA.
