Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions.

Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Research Abstract Details 

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Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Abstract Text:

Maxim Berezovski,Sergey N Krylov,

We introduce a novel electrophoretic method, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), and demonstrate its use for studying protein-DNA interactions. The equilibrium mixture of protein and DNA contains three components: free protein, free DNA, and the protein-DNA complex. A short plug of such a mixture is injected into the capillary, and the three components are separated under nonequilibrium conditions. The resulting electropherograms are composed of characteristic peaks and exponential curves. An easy nonnumerical analysis of a single electropherogram reveals two parameters: the equilibrium binding constant and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the product of the two experimentally determined constants. NECEEM was applied to study the interaction between single-stranded DNA binding protein and a fluorescently labeled 15-mer oligonucleotide. It allowed us to measure for the first time the rate constant of complex decay for this important protein-DNA pair, k-1 = 0.03 s-1. The value of the equilibrium binding constant, Kb = 3.6 x 10-6 M-1, was in good agreement with those measured by other methods. As low as 10-18 mol of the protein was sufficient for the measurements. Thus, the new method is simple, informative, and highly sensitive. Moreover, it can be equally applied to other noncovalent protein-ligand complexes. These features of NECEEM make this method an indispensable tool in studies of macromolecular interactions. They also emphasize the potential role of NECEEM in the development of extremely sensitive protein assays using nucleotide aptamers.

Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Publishing Authors By Initials

M Berezovski,SN Krylov,

For similar polynucleotides: oligonucleotides research abstracts see: polynucleotides: oligonucleotides research

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Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of the American Chemical Society

VOLUME: 124

Page Numbers: 13674-5

Journal Abbreviation: J. Am. Chem. Soc.

ISSN: 0002-7863

DAY: 20

MONTH: Nov

YEAR: 2002

Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Information

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LANGUAGE: eng

NlmUniqueID: 7503056

Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Keywords Mesh Terms:

KEYWORDS: Oligonucleotides

MESH TERMS: metabolism

Chemical & Substance for Abstract: Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Information

Substance Name: DNA

Registry Number: 9007-49-2

Grant and Affiliation Information for Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions.

AFFILIATION: Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.

Country: United States

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MEDLINETA: J Am Chem Soc

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