Effect of Hydrogen Ion Concentration on the Absorption Spectrum and Fluorescence Life Time of Chloroplasts

Photosynthesis, Chlorophyll, Chloroplasts, Fluorescence Life Time, Circular Dichroism Subjecting pea chloroplasts to hydrogen ion gradients results in small but significant changes in the absorption and circular dichroic spectra. The relative intensities of the fast and slow fluorescence decays are modified by the hydrogen ion concentration. The life time of the slow components is only slightly altered. At pH 11 the circular dichroic peak at 690 nm, assigned to Photosystem I chlorophyll-protein complex, is clearly resolved. Also, there is a splitting of the peak at 650-640 nm, attributed to Photosystem II chlorophyll-protein. At pH 3.9 splitting of the 650-640 nm peak is observed. There is also a positive component at 705 nm which may be associated with an aggregated form of chlorophyll or pheophytin. The changes in absorption spectra are determined by measuring the difference in spectra between identical chloroplasts suspensions one at pH 7.6 and the other at alkaline or acidic pH. At pH 2 and pH 3.7 there are time dependent increases in absorbance at 430, 445, 520, 538, 670 nm, and a decrease in absorbance at 606 nm. After a delay of 11 min at pH 2, there is also an increase in absorbance at 700 nm. The latter is interpreted as possible formation of aggregated pheophytin. At pH 11.0 there are time dependent increases in absorbance at 638, 454 and 440 nm, and decreases in absorbance at 675, 660, 508 and 472 nm. The changes in absorbance of the pigment around 520 nm (associated with an electrochromic effect) is interpreted as resulting from a leakage of hydrogen ion across the thylakoid membrane. The spectral changes that occur during the first 9 min are reversible. After the chloroplasts have incubated for more than 15 min most of the spectral changes are not reversible.