Novel Fluorescently-labeled Enzyme Substrates for the Sensitive Detection of HIV-protease

摘要

In this paper we applied the efficient fluorescence quenching of the red-absorbing oxazine derivative MR121 by theamino acid tryptophan to develop a new fluorescence based enzyme assay that can be used for detection of exopeptidasesand endopeptidases. Therefore, we developed peptide substrates labeled with only one chromophore, which is quenchedby a neighbored tryptophan residue via photoinduced electron transfer. The specific cleavage site for the target enzyme islocated between the chromophore and the tryptophan residue. After digestion of the substrate the contact formationbetween tryptophan and fluorescent dye is precluded and a significant increase in fluorescence intensity occurs. Todemonstrate the new assay technique for exopeptidases, a substrate for the Carboxypeptidase A was designed and adetection limit below the picomolar range (~10-13 M) was achieved with standard fluorescence spectrometry. Theprimary objective was the detection of the HIV-protease, which is an endopeptidase digesting substrates containingseven specific amino acids in the cleavage site. We designed a substrate, which enables the detection of 10-9 M HIVprotease,whereas the continuous monitoring of the fluorescence signal also allows kinetic studies.