Determination of Intrathylakoid pH from the Exchange Broadening of the ESR Spectrum of TEMPOamine Spin Label in Chloroplasts

The study is devoted to the substantiation of the method for measuring the transmembrane pH difference (ΔpH) in chloroplasts. The method is based on the phenomenon of concentration-dependent broadening of the ESR spectrum of TEMPOamine spin label (TA) molecules, which accumulate inside thylakoids in response to the photoinduced acidification of intrathylakoid space. The study of the concentration dependence of the ESR spectra of TA molecules localized in the aqueous phase of intrathylakoid space indicated that a marked spectrum broadening, which is due to the exchange interaction of nitroxyl radicals, makes itself evident only after the threshold concentration of the spin label is reached [TA]ⁱⁿ_θ ≅ 2,5-3,5 MM. With a knowledge of [TA]ⁱⁿ_θ, ΔpH in chloroplasts can be quantified. To do this, it is necessary to find a concentrations of TA in external medium ([TA]*_out), above which the concentration of TA molecules that accumulate inside thylakoids will exceed the threshold level [TA]_in=[TA]ⁱⁿ_θ and therefore the absorption of TA by thylakoids can be recorded from the effect of the photoinduced broadening of the ESR spectrum. The [TA]*_out value can be estimated from the dependence of the photoinduced broadening of the spectrum on the concentration of TA in the culture medium of chloroplasts after which the value ΔpH=lg ([TA]_θ/[TA]*_out) can be calculated.