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

Gerdes, B.; Rixen, R. M.; Kramer, K.; Forbrig, E.; Hildebrandt, P.; Steinem, Claudia

doi:10.1007/s00216-018-1248-7

Item

ITEM ACTIONSEXPORT

Add to Basket

Local TagsRelease HistoryDetailsSummary

Released

Journal Article

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

MPS-Authors

/persons/resource/persons220764

Steinem, Claudia
Max Planck Fellow Group Membrane-based biomimetic nano- and micro-compartments, Max Planck Institute for Dynamics and Self-Organization, Max Planck Society;

External Resource

No external resources are shared

Fulltext (restricted access)

There are currently no full texts shared for your IP range.

Fulltext (public)

There are no public fulltexts stored in PuRe

Supplementary Material (public)

There is no public supplementary material available

Citation

Gerdes, B., Rixen, R. M., Kramer, K., Forbrig, E., Hildebrandt, P., & Steinem, C. (2018). Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay. Analytical and Bioanalytical Chemistry, 410(25), 6497-6505. doi:10.1007/s00216-018-1248-7.

Cite as: https://hdl.handle.net/21.11116/0000-0001-DE33-C

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 ᅟ.