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Analytical and Bioanalytical Chemistry

, Volume 410, Issue 25, pp 6497–6505 | Cite as

Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay

  • Benjamin Gerdes
  • Rebecca M. Rixen
  • Kristina Kramer
  • Enrico Forbrig
  • Peter Hildebrandt
  • Claudia Steinem
Research Paper

Abstract

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H+-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the KD-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters.

Graphical abstract

Keywords

ACMA assay Lipid-coupled fluorophore pH-sensitive dye Proton channel SEIRA 

Notes

Acknowledgements

We thank Prof. Dr. T. Friedrich for the Hv1 cDNA. We thank J. Gerber-Nolte for technical support.

Funding information

This study received financial support from the VW-foundation and the DFG (EXC314).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1248_MOESM1_ESM.pdf (834 kb)
ESM 1 (PDF 833 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institut für Organische und Biomolekulare ChemieUniversität GöttingenGöttingenGermany
  2. 2.Institut für ChemieTechnische Universität BerlinBerlinGermany
  3. 3.Max-Planck-Institut für Dynamik und SelbstorganisationGöttingenGermany

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