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. 1988 Apr;86(4):1257–1263. doi: 10.1104/pp.86.4.1257

Quantitative and Rapid Estimation of H+ Fluxes in Membrane Vesicles 1

Software for Analysis of Fluorescence Quenching and Relaxation

Ian R Jennings 1,2, Philip A Rea 1,2,2, Roger A Leigh 1,2, Dale Sanders 1,2
PMCID: PMC1054661  PMID: 16666064

Abstract

Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na+/H+ antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na+ -dependent fluorescence changes are derived and can be used for Km determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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