Methods for determining the reduction potentials of flavin enzymes

Shelbi L. Christgen, Sophia M. Becker, Donald F Becker

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

A key factor for flavoenzyme activity is the reduction potential of the bound flavin. The reduction potentials of protein-bound flavins span approximately a 500-mV range consistent with flavoenzymes having critical roles in metabolism and a variety of biological processes. Redox potentials of flavoenzymes have traditionally been determined using an electrode-based system with either direct or indirect electrochemical coupling between the protein and the working electrode. An electrode independent method, however, is also now commonly used and involves calculating the unknown flavin reduction potential of the protein from the known reduction potential of a reference or indicator dye. Here, the “classic” potentiometric method and the xanthine/xanthine oxidase methods are described. Both methods rely on equilibrium between protein-bound flavin and redox dyes. The potentiometric method measures the equilibrated redox potential of the protein-dye mixture whereas the xanthine/xanthine oxidase technique relies on slow continuous enzymatic reduction to maintain a constant equilibrium between the protein and the dyes. Because electrochemical equipment is not required, the xanthine/xanthine oxidase method is more accessible and convenient for researchers seeking to determine reduction potentials of flavoproteins or other biological redox centers such as hemes. The xanthine/xanthine oxidase method has been used to determine flavin reduction potentials from + 132 to − 417 mV, demonstrating it is suitable for characterizing the redox properties of most flavoproteins.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

Xanthine
Xanthine Oxidase
Oxidation-Reduction
Enzymes
Coloring Agents
Flavoproteins
Electrodes
Proteins
Flavins
Biological Phenomena
Equilibrium constants
4,6-dinitro-o-cresol
Heme
Metabolism
Research Personnel
Equipment and Supplies

Keywords

  • Flavin
  • Flavoenzyme
  • Potentiometric titration
  • Reduction potential
  • Xanthine
  • Xanthine oxidase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Christgen, S. L., Becker, S. M., & Becker, D. F. (2019). Methods for determining the reduction potentials of flavin enzymes. In Methods in Enzymology (Methods in Enzymology). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2019.03.004

Methods for determining the reduction potentials of flavin enzymes. / Christgen, Shelbi L.; Becker, Sophia M.; Becker, Donald F.

Methods in Enzymology. Academic Press Inc., 2019. (Methods in Enzymology).

Research output: Chapter in Book/Report/Conference proceedingChapter

Christgen, SL, Becker, SM & Becker, DF 2019, Methods for determining the reduction potentials of flavin enzymes. in Methods in Enzymology. Methods in Enzymology, Academic Press Inc. https://doi.org/10.1016/bs.mie.2019.03.004
Christgen SL, Becker SM, Becker DF. Methods for determining the reduction potentials of flavin enzymes. In Methods in Enzymology. Academic Press Inc. 2019. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2019.03.004
Christgen, Shelbi L. ; Becker, Sophia M. ; Becker, Donald F. / Methods for determining the reduction potentials of flavin enzymes. Methods in Enzymology. Academic Press Inc., 2019. (Methods in Enzymology).
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