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Membrane capacitance; Phosphatidylcholine; Protonation; Time resolution
Abstract:
The laser-induced proton pulse (Gutman, M. (1986) Methods Enzymol. 127, 522-538) was used for transient protonation of one side of a black lipid membrane. The charging of the membrane drives an electric (voltage or current) signal selectively representing the fast proton exchange at the membrane/electrolyte interface. The sensitivity of the electric signal to the presence of buffer indicates that proton transfer is measured, not some dyes or membrane photoelectric artifact. The same event can be visualized in an analogous system consisting of a pH indicator adsorbed to neutral detergent-phospholipid mixed micelles. The time-resolved light absorption transient is equivalent to the electrically determined transient charging of the membrane surface. The sensitivity of the current measurement exceeds the spectrophotometric method by 6-8 orders of magnitudes. As little as 10-18 mol of H+ reacting with 0.75 mm2 of the membrane surface can be monitored in a time-resolved observation. Both types of observed transients were accurately reconstructed by the numerical solution of coupled, non-linear, differential equations describing the system. The rate constants of the various proton transfer reactions were calculated and found to be of diffusion controlled reactions. There is no evidence for any barrier at the interface which either prevents protons from reaching the membrane, or keeps proton on the interface. The electric measurements can be applied for monitoring proton transfer kinetics of complex biomembrane preparations.
