DETERMINATION OF INTRATHYLAKOID PH FROM T HE 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 (Delta pH) in chloroplasts. The metho d is based on the phenomenon of concentration-dependent broadening of the ESR spectrum of TEMPOamine spin label (TA) molecules, which accumu late inside thylakoids in response to the photoinduced acidification o f intrathylakoid space. The study of the concentration dependence of t he ESR spectra of TA molecules localized in the aqueous phase of intra thylakoid space indicated that a marked spectrum broadening, which is due to the exchange interaction of nitroxyl radicals, makes itself evi dent only after the threshold concentration of the spin label is reach ed [TA](theta)(in) congruent to 2,5-3,5 mM. With a knowledge of [TA](t heta)(in), Delta pH in chloroplasts can be quantified. To do this, it is necessary to find a concentrations of TA in external medium ([TA](o ut)), above which the concentration of TA molecules that accumulate i nside thylakoids will exceed the threshold level [TA](in)=[TA](theta)( in) and therefore the absorption of TA by thylakoids can be recorded f rom the effect of the photoinduced broadening of the ESR spectrum. The [TA](out) value can be estimated from the dependence of the photoind uced broadening of the spectrum on the concentration of TA in the cult ure medium of chloroplasts after which the value Delta pH=1g ([TA](the ta)/[TA](out)) can be calculated.