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A theoretical approach to some analytical properties of heterogeneous enzymatic assays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics. (CEP. University of Havana.)ORCID iD: 0000-0003-3522-0196
2004 (English)In: Analytical Chemistry, Vol. 76, 2664-2668 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2004. Vol. 76, 2664-2668 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94503OAI: oai:DiVA.org:uu-94503DiVA: diva2:168373
Available from: 2006-05-08 Created: 2006-05-08 Last updated: 2015-03-01
In thesis
1. Sensitivity, Noise and Detection of Enzyme Inhibition in Progress Curves
Open this publication in new window or tab >>Sensitivity, Noise and Detection of Enzyme Inhibition in Progress Curves
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Starting with the development of an enzymatic assay, where an enzyme in solution hydrolysed a solid-phase bound peptide, a model for the kinetics of enzyme action was introduced. This model allowed the estimation of kinetic parameters and enzyme activity for a system that has the peculiarity of not being saturable with the substrate, but with the enzyme. In a derivation of the model, it was found that the sensitivity of the signal to variations in the enzyme concentration had a transient increase along the reaction progress with a maximum at high substrate conversion levels.

The same behaviour was derived for the sensitivity in classical homogeneous enzymatic assays and experimental evidence of this was obtained. The impact of the transient increase of the sensitivity on the error structure, and on the ability of homogeneous end-point enzymatic assays to detect competitive inhibition, came into focus. First, a non-monotonous shape in the standard deviation of progress curve data was found and it was attributed to the random dispersion in the enzyme concentration operating through the transient increase in the sensitivity. Second, a model for the detection limit of the quantity Ki/[I] (the IDL-factor) as a function of the substrate conversion level was developed for homogeneous end-point enzymatic assays.

It was found that the substrate conversion level where the IDL-factor reached an optimum was beyond the initial velocity range. Moreover, at this optimal point not only the ability to detect inhibitors but also the robustness of the assays was maximized. These results may prove to be relevant in drug discovery for optimising end point homogeneous enzymatic assays that are used to find inhibitors against a target enzyme in compound libraries, which are usually big (>10000) and crowded with irrelevant compounds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 184
Keyword
Biochemistry, Progress curves, limit of detection, Z'-factor, primary screening, enzyme inhibition, error structure, sensitivity analysis, High-throughput screning, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6886 (URN)91-554-6569-2 (ISBN)
Public defence
2006-05-29, Room B41, BMC, Husarg. 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2006-05-08 Created: 2006-05-08 Last updated: 2017-05-04Bibliographically approved

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Gutierrez Arenas, Omar

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