Real-time colorimetric screening of endopeptidase inhibitors using adenosine triphosphate (ATP)-stabilized gold nanoparticles

The gold nanoparticles (AuNPs) that were stabilized with adenosine triphosphate (ATP) were stable over a wide range of pHs for the buffer, even in the presence of high concentrations of salt and protein. However, these stabilized AuNPs immediately aggregated when they were exposed to thiol-containing compounds, such as thiophenol. Endoprotease hydrolyzed the thioester bond in the CBZ–Phe–S–Ph substrate, and the hydrolyzed product (thiophenol) reacted with the AuNPs that were stabilized with ATP, causing them to aggregate, which in turn resulted in a visible color change in the AuNPs solution. This method enabled the real-time monitoring of the inhibition potencies of various endopeptidase inhibitors and the activity of endoprotease. This assay discriminated between the inhibition activities of various protease inhibitors for endoprotease on the basis of the color change of the assay solution.