ArticleAnalytical biochemistry2017
Acquisition of accurate data from intramolecular quenched fluorescence protease assays.
Article in Analytical biochemistry, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- Green Synthesis of Graphene Quantum Dots (GQDs) and Carbon Dots (CDs) Mediated with Erythrina caffra for Potential Antiviral Properties Against SARS-CoV-2.Materials (Basel, Switzerland) · 2026Article
- Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology.Journal of molecular biology · 2020Review
- The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.Proteins · 2020Article
- Phosphoramidon inhibits the integral membrane protein zinc metalloprotease ZMPSTE24.Acta crystallographica. Section D, Structural biology · 2018Article
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2 authors.
Funding
Abstract
The Intramolecular Quenched Fluorescence (IQF) protease assay utilizes peptide substrates containing donor-quencher pairs that flank the scissile bond. Following protease cleavage, the dequenched donor emission of the product is subsequently measured. Inspection of the IQF literature indicates that rigorous treatment of systematic errors in observed fluorescence arising from inner-filter absorbance (IF) and non-specific intermolecular quenching (NSQ) is incompletely performed. As substrate and product concentrations vary during the time-course of enzyme activity, iterative solution of the kinetic rate equations is, generally, required to obtain the proper time-dependent correction to the initial velocity fluorescence data. Here, we demonstrate that, if the IQF assay is performed under conditions where IF and NSQ are approximately constant during the measurement of initial velocity for a given initial substrate concentration, then a simple correction as a function of initial substrate concentration can be derived and utilized to obtain accurate initial velocity data for analysis.
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