Skip Navigation
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

National Institute of Environmental Health Sciences

Use this QR code to view the newest version of this document
Use this QR code to view the newest version of this document

Your Environment. Your Health.

Publication Detail

Title: Tryptophan fluorescence quenching by enzyme inhibitors as a tool for enzyme active site structure investigation: epoxide hydrolase.

Authors: Matveeva, Evgenia G; Morisseau, Christophe; Goodrow, Marvin H; Mullin, Chris; Hammock, Bruce D

Published In Curr Pharm Biotechnol, (2009 Sep)

Abstract: We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtol-urea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH +XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)(SV). Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)(SV) versus inhibiting strength, IC(50) values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC(50) values for several highly potent inhibitors, with IC(50) values at the limit of the IC(50) detection sensitivity by colorimetric enzyme assay. IC(50) values determined from our quenching assay show correlation with IC(50) values determined in the literature by more sensitive radioactive-based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.

PubMed ID: 19619123 Exiting the NIEHS site

MeSH Terms: Binding Sites; Enzyme Activation; Enzyme Inhibitors/chemistry*; Epoxide Hydrolases/chemistry*; Protein Binding; Spectrometry, Fluorescence/methods*; Tryptophan/chemistry*

Back
to Top