Temperature jump as a new technique to study the kinetics of fast transport of protons across membranes

Abstract

Application of a temperature jump (2.5 ° C) to a suspension of liposomes, having phosphate (ΔpK/ΔT approximately 0.005) as the internal buffer and tris(hydroxymethyl)aminomethane (ΔpK/ΔT approximately 0.031) as the external buffer, created a ΔpH (pH_in - pH_out) of positive sign in ca. 5 μs. Decay of this ΔpH was monitored by using the fluorescent pH indicator 8-hydroxy-1,3,6-pyrenetrisulfonic acid entrapped inside the liposome. This technique is useful to study transmembrane proton movement in the time range 5 μs-10 s at physiological pH values. The kinetics of proton transport aided by ion carriers such as nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin were studied by our method. The electrogenic nature of transport by CCCP and valinomycin and electroneutral ion transport by nigericin and monensin were shown. From the kinetics of proton transport aided by gramicidin, the time-averaged single-channel conductance of gramicidin channels was estimated to be (2.1 ± 0.5) × 10^-16 S for H⁺ at pH 7.5.