Determination of a transmembrane pH difference in chloroplasts with a spin label tempamine

Abstract

We present a method for measuring the transmembrane pH difference (ΔpH=pH in−pH out) in chloroplasts with a spin label TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl) accumulating inside the thylakoids in response to generation of ΔpH. Experiments with chloroplasts suspended in the media of different osmolarity demonstrated that most of TEMPAMINE (TA) molecules taken up by chloroplasts were localized in the bulk of the thylakoid lumen. The ΔpH value was determined from the relationship ΔpH=lg([H +] in/[H +] out)≅lg( C in/ C out), where C in and C out are the concentrations of TA inside and outside the thylakoids, respectively. To quantify the internal concentration C in, we used the threshold nature of the concentration-dependent broadening of the EPR signal from TA. It was demonstrated that spin-exchange interactions between TA molecules caused an observable broadening of the signal only when the concentration of TA exceeded the threshold level, [ TA] ϑ≈2.0–2.2 mM . The concentration dependencies of the signal parameters (the peak-to-peak amplitude, A pp, and the linewidth, Δ H pp) were described within a model of the non-homogeneous broadening of an unresolved hyperfine multiplet from the protons of TA molecule. If the concentration of TA inside the thylakoids went beyond the threshold level, the spin-exchange broadening of the EPR signal was accompanied by a reversible decrease in the signal height (parameter Δ A). By measuring the signal behavior at different levels of microwave power, we were able to discriminate between the line broadening effects caused by concentrating TA molecules inside the thylakoids or the light-induced changes in the concentration of oxygen. We developed a general algorithm for determination of the ΔpH value and the internal volume of thylakoids, V in, from the non-linear dependence of parameter Δ A on the concentration C 0 of TA in a chloroplast suspension. Advantages of this method are: (i) it avoids the use of a broadening agent; (ii) it allows the internal volume of thylakoids to be evaluated; and (iii) the concentrations of TA used to measure the ΔpH are below the range of concentrations that could cause the uncoupling electron transport to ATP synthesis in chloroplasts. Results of our measurements are consistent with the literature data on ΔpH determinations by other methods.