4 April 2005 Novel fluorescently labeled enzyme substrates for the sensitive detection of HIV-protease
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In this paper we applied the efficient fluorescence quenching of the red-absorbing oxazine derivative MR121 by the amino acid tryptophan to develop a new fluorescence based enzyme assay that can be used for detection of exopeptidases and endopeptidases. Therefore, we developed peptide substrates labeled with only one chromophore, which is quenched by a neighbored tryptophan residue via photoinduced electron transfer. The specific cleavage site for the target enzyme is located between the chromophore and the tryptophan residue. After digestion of the substrate the contact formation between tryptophan and fluorescent dye is precluded and a significant increase in fluorescence intensity occurs. To demonstrate the new assay technique for exopeptidases, a substrate for the Carboxypeptidase A was designed and a detection limit below the picomolar range (~10-13 M) was achieved with standard fluorescence spectrometry. The primary objective was the detection of the HIV-protease, which is an endopeptidase digesting substrates containing seven specific amino acids in the cleavage site. We designed a substrate, which enables the detection of 10-9 M HIV-protease, whereas the continuous monitoring of the fluorescence signal also allows kinetic studies.
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Thorsten Martin Staudt, Thorsten Martin Staudt, Lutz Knemeyer, Lutz Knemeyer, Hans-Georg Krausslich, Hans-Georg Krausslich, Jens-Peter Knemeyer, Jens-Peter Knemeyer, Nicole Marme, Nicole Marme, "Novel fluorescently labeled enzyme substrates for the sensitive detection of HIV-protease", Proc. SPIE 5704, Genetically Engineered and Optical Probes for Biomedical Applications III, (4 April 2005); doi: 10.1117/12.589101; https://doi.org/10.1117/12.589101

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