title: Determination of concentration and activity of immobilized enzymes.
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.
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Holly
authors.initials: H
authors.lastname: Morris
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Tivanski
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA. Electronic address: amnon-kohen@uiowa.edu.
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
mesh_terms: D020134:Catalytic Domain; D057075:Enzyme Assays / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D013762:Tetrahydrofolate Dehydrogenase
keywords: Activity assay; Dihydrofolate reductase; Immobilization; Radiolabeled inhibitor