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| Titel: |
New HIV-protease assays applying self-quenching peptide substrates in combination with time-resolved fluorescence single-molecule spectroscopy |
| Auteur: |
Staudt, Thorsten Martin Krausslich, Hans-Georg Knemeyer, Jens-Peter Marme, Nicole |
| Verschenen in: |
International journal of environmental analytical chemistry |
| Paginering: | Jaargang 87 (2007) nr. 10-11 pagina's 731-743 |
| Jaar: |
2007-08 |
| Inhoud: |
This work describes the optimization and adoption of an assay system for the Human Immunodeficiency Virus (HIV)-protease, whose inhibition plays a central role in HIV therapy. The HIV-protease, which is an essential enzyme during viral maturation, has a specific cleavage site of eight amino acid residues (SQNY*PIV). Adding two amino acid residues at the N-terminus and enclosing the resulting sequence by a dye-labelled lysine residue and a tryptophan residue leads to the substrate (K(dye)CGSQNY*PIVW) in which the fluorescence of the fluorophore is efficiently quenched by the intrinsic tryptophan due to a photoinduced electron transfer reaction. After cleavage of the substrate by the target enzyme, the dye and the tryptophan residue are separated, effecting a significant increase in fluorescence intensity. Measuring the fluorescence versus time enables an online-monitoring of the enzyme activity. With this method, a HIV-PR concentration of 10-9 M is detectable within minutes, which is comparable with commercially available assays using doubly labelled substrates based on a fluorescence resonance energy transfer. We were able to further increase the sensitivity to the subnanomolar range by using confocal single-molecule spectroscopy. |
| Uitgever: |
Taylor & Francis |
| Bronbestand: |
Elektronische Wetenschappelijke Tijdschriften |
Details van artikel 9 van 11 gevonden artikelen
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