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How do protons cross the membrane-solution interface? Kinetic studies on bilayer membranes exposed to the protonophore S-13 (5-chloro-3-tert-butyl-2′-chloro-4′ nitrosalicylanilide)

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Summary

A simple carrier model describes adequately the transport of protons across lipid bilayer membranes by the weak acid S-13. We determined the adsorption coefficients of the anionic, A−, and neutral, HA, forms of the weak acid and the rate constants for the movement of A− and HA across the membrane by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. These measurements agree with the results of voltage clamp and charge pulse kinetic experiments. We considered three mechanisms by which protons can cross the membranesolution interface. An anion adsorbed to the interface can be protonated by (i) a H+ ion in the aqueous phase (protolysis), (ii) a buffer molecule in the aqueous phase or (iii) water molecules (hydrolysis). We demonstrated that the first reaction cannot provide the required flux of protons: the rate at which H+ must combine with the adsorbed anions is greater than the rate at which diffusion-limited reactions occur in the bulk aqueous phase. We also ruled out the possibility that the buffer is the main source of protons: the rate at which buffers must combine with the adsorbed anions is greater than the diffusion-limited rate when we reduced the concentration of polyanionic buffer adjacent to the membrane-solution interface by using membranes with a negative surface charge. A simple analysis demonstrates that a hydrolysis reaction can account for the kinetic data. Experiments at acid pH demonstrate that the transfer of H+ from the membrane to the aqueous phase is limited by the rate at which OH combines with adsorbed HA and that the diffusion coefficient of OH− in the water adjacent to the bilayer has a value characteristic of bulk water. Our experimental results demonstrate that protons are capable of moving rapidly across the membrane-solution interface, which argues against some mechanisms of local chemiosmosis.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, State University of New York, 11794, Stony Brook, New York

    John Kasianowicz & Stuart McLaughlin

  2. Lehrstuhl für Biotechnologie, Universität Würzburg, D-8700, Würzburg, Federal Republic of Germany

    Roland Benz

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  1. John Kasianowicz
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  2. Roland Benz
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  3. Stuart McLaughlin
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Kasianowicz, J., Benz, R. & McLaughlin, S. How do protons cross the membrane-solution interface? Kinetic studies on bilayer membranes exposed to the protonophore S-13 (5-chloro-3-tert-butyl-2′-chloro-4′ nitrosalicylanilide). J. Membrain Biol. 95, 73–89 (1987). https://doi.org/10.1007/BF01869632

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  • DOI: https://doi.org/10.1007/BF01869632

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