The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent K_m = 4 μM and saturation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is approx. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.