Measurement of chloroplast internal protons with 9-aminoacridine. Probe binding, dark proton gradient, and salt effects

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A defined ratio, γ, of the total proton uptake to the concentration change of free internal H + is observed for illuminated envelope-free chloroplasts (Haraux, F. and de Kouchkovsky, Y. (1979) Biochim. Biophys. Acta, 546, 455–471). Proton uptake is measured by the external pH shift, free internal H + by 9-aminoacridine fluorescence quenching. Extension of this work leads to the following conclusions, which, in the case of 9-aminoacridine behaviour, should apply to any kind of diffusible protonizable ΔpH probe: 1. 1. The γ constancy is preserved when the internal volume ( V i) is modulated by chlorophyll and osmolarity changes: thus, 9-aminoacridine behaves as expected from the ΔpH distribution of an amine of high p K; previous doubts on this point are attributed to the lack of control of the external proton uptake. 2. 2. With variable 9-aminoacridine concentration, however, some variation of γ confirms the existence of slight light-induced probe-membrane interactions. 3. 3. According to the diffuse layer theory, salts decrease the negative potential at the ‘plane of closest approach’ of the thylakoids, thereby releasing the excess 9-aminoacridine in this diffuse layer, which increases its fluorescence. Although of equal valency, NH + 4 is more potent than K +, suggesting competition between amines for specific anionic binding sites. 4. 4. Two categories of membrane modifications are induced by salts: in addition to the above-mentioned electrical effect, mono- and divalent cations at high concentration increase the chloroplast proton binding capacity. La 3+ is only able to release the excess dye in the diffuse layer and leaves γ unchanged. Therefore the probe-membrane interactions should have limited importance for steady-state ΔpH measurement. 5. 5. A Donnan-type dark pH difference, which could seriously bias these ΔpH estimates, is found experimentally to be less than 2 (no significant γ change when V i varies) and even theoretically less than 1 (on the basis of the concentration of the non-diffusible internal protonizable groups). Similarly, the predictable errors of V i and its possible light-induced variations must have a small effect on ΔpH under present experimental conditions.