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

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.

Extending the working range of immunoanalysis by exploitation of two monoclonal antibodies.

A newly established rat monoclonal antibody (mAb) for isoproturon, namely, IOC 10G7, is described. This mAb shows a standard curve for isoproturon in phosphate-buffered saline with a test midpoint of 5.5 +/- 1.

Enzyme-linked immunosorbent assays based on rabbit polyclonal and rat monoclonal antibodies against isoproturon.

This work describes the production and characterization of rabbit polyclonal antisera (pAb) and rat monoclonal antibodies (mAb) against isoproturon. Coating antigen and enzyme-tracer formats were developed. Standard curves for isoproturon were conducted either in 40 mM phosphate buffered saline (PBS) or in Milli-Q water.

Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae).

A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N'-propylurea and N-hexadecyl-N'-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC(50) around 90 nM.

Structural refinement of inhibitors of urea-based soluble epoxide hydrolases.

The soluble epoxide hydrolase (sEH) is involved in the metabolism of arachidonic, linoleic, and other fatty acid epoxides, endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas, carbamates, and amides are new potent and stable inhibitors of sEH. However, the poor solubility of the lead compounds limits their use.