Determination of concentration and activity of immobilized enzymes

Priyanka Singh; Holly Morris; Alexei V Tivanski; Amnon Kohen

doi:10.1016/j.ab.2015.02.014

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Determination of concentration and activity of immobilized enzymes

Journal article

Peer reviewed

Determination of concentration and activity of immobilized enzymes

Priyanka Singh, Holly Morris, Alexei V Tivanski and Amnon Kohen

Analytical biochemistry, Vol.484, pp.169-172

09/01/2015

DOI: 10.1016/j.ab.2015.02.014

PMCID: PMC4497861

PMID: 25707319

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https://www.ncbi.nlm.nih.gov/pmc/articles/4497861View

Open Access

Abstract

Methods that directly measure the concentration of surface-immobilized biomolecules are scarce. More commonly, the concentration of the soluble molecule is measured before and after immobilization, and the bound concentration is assessed by elimination, assuming that all bound molecules are active. An assay was developed for measuring the active site concentration, activity, and thereby the catalytic turnover rate (kcat) of an immobilized dihydrofolate reductase as a model system. The new method yielded a similar first-order rate constant, kcat, to that of the same enzyme in solution. The findings indicate that the activity of the immobilized enzyme, when separated from the surface by the DNA spacers, has not been altered. In addition, a new immobilization method that leads to solution-like activity of the enzyme on the surface is described. The approaches developed here for immobilization and for determining the concentration of an immobilized enzyme are general and can be extended to other enzymes, receptors, and antibodies.

Catalytic Domain

Enzyme Assays - methods

Enzymes, Immobilized - chemistry

Enzymes, Immobilized - metabolism

Escherichia coli - enzymology

Kinetics

Models, Molecular

Tetrahydrofolate Dehydrogenase - chemistry

Tetrahydrofolate Dehydrogenase - metabolism

Details

Title: Subtitle: Determination of concentration and activity of immobilized enzymes
Creators: Priyanka Singh - Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA
Holly Morris - Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA
Alexei V Tivanski - Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA
Amnon Kohen - Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA. Electronic address: amnon-kohen@uiowa.edu
Resource Type: Journal article
Publication Details: Analytical biochemistry, Vol.484, pp.169-172
DOI: 10.1016/j.ab.2015.02.014
PMID: 25707319
PMCID: PMC4497861
NLM abbreviation: Anal Biochem
ISSN: 0003-2697
eISSN: 1096-0309
Grant note: GM65368 / NIGMS NIH HHS R01 GM065368 / NIGMS NIH HHS
Language: English
Date published: 09/01/2015
Academic Unit: Chemistry
Record Identifier: 9984216689402771

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