ANALYSE DER PHOTOSYNTHESE MIT BLITZLICHT

Abstract

The primary reactions of photosynthesis are fast reactions. To get detailed informations we developed three different methods. 1. sensitive flash photometry 38, 2. periodically chemical relaxation51, 26a, 3. manometry in flashing light groups 26b. With the method of flash photometry fast absorption changes in suspensions of chlorella and spinach chloroplasts were studied. 7 different types of absorption changes have been separated and analysed (Fig. 2). Older results 28-49lead to a reaction scheme published in l. c. 13. This scheme was refined (s. Fig. 3) by results published in l. c. 14-26. In the following 6 papers these investigations are described in detail and supplement by new results. Separation of the difference-spectrum of chlorophyll-al (P 700) in 5 ways. Under “normal” conditions (exciting Hill - active chloroplasts with blue light between 380 and 480 mμ or with red light between 620 and 720 mµ) mixed changes of absorption can be observed between 400 and 800 mµ (Fig. 4, this difference-spectrum does not include the changes with τ<10-3 sec, s. Fig. 2). Out of this overall difference-spectrum one component with changes of absorption at 430 and 703 mμ could be separated by using the following different systems: a) aged chloroplasts reactivated by addition of reduced DPIP or reduced PMS (ascorbate in excess) (Fig. 5), b) plastoquinone-extracted chloroplasts [extraction with petroleumether] (Fig. 6), c) digitonin-treated chloroplasts reactivated by addition of reduced PMS [ascorbate in excess] (Fig. 7), d) chloroplasts at -150°C with addition of reduced PMS [ascorbate in excess] (Fig. 8), e) chloroplasts under the influence of far-red background-light [728 mµ] (Fig. 9). Kinetics. During the flash the absorption at 430 and 703 mµ decreases very fast (<10-5 sec). In the dark the back reaction takes place in ≈10-2 sec at 20°C (s. Fig. 10). This reaction time is always observable in the presents of far redbackground light λ>700 mμ. (The detecting light at 703 mµ can act already as far red background light.) At very low itensities of far red background light the backreaction takes place however in <10-4 sec (details s. l. c. 25a). Identification of chlorophyll-al. The upper results (5 equal spectra under different conditions) suggest that the changes of absorption at 430 and 703 mμ are caused by one substance. This was additionally proved by comparing the magnitude and the kinetic of both changes in reactivated aged or digitonin-treated chloroplasts under different conditions. The ratio of the amplitudes and the halflifes are identical at 430 and 703 mµ at different values of pH (Fig. 11 and Fig. 12) and also at different concentrations of added reduced PMS (Fig. 13). Decreases of absorption just within the two absorption bands of chlorophyll-a indicate that very probably a chlorophyll-a (Chl-aI-430-703) is in action. From the magnitude of the changes of absorption at 703 mμ it follows, that Chl-al has a concentration of 0,1% of the bulk of chlorophyll. Oxidation of Chl-ai. The decreases of absorption indicate an oxidation of Chl-al in the light. This is proved by the fact, that in aged or digitonin-treated chloroplasts reduced PMS can be directly coupled to the light product. This is demonstrated by the strong acceleration of the back reaction with increasing concentrations of reduced PMS (Fig. 13 and Fig. 15) and by the demonstration of a first order back reaction (Fig. 14 and Fig. 15). The experimental results are theoretically explained. It is proved, that the light product Chl-aI⨁ reacts with PMSH⊖ [compare measuring points and theoretical curve in Fig. 12] [s. scheme (1)]. From the measurements it follows a reaction constant of 1,5·107 l/Mol·sec (Fig. 15) and an energy of activation of 3,8 kcal/Mol (Fig. 16) for the reaction of Chl-aI⨁ with PMSH⊖. Chl-aI-oxidation as a primary act. The fact, that Chl-ai⊕ is built up within <10-5 sec (20°C) [Fig. 10] and that Chl-aI⨂ could be trapped at -150°C (Fig. 8 and Fig. 17) give evidence, that this oxidation is a primary act. The electron acceptor of Chl-ai is called Ζ. Ζ⊖ reduces via intermediary products TPN. Under the reported conditions a - e one light reaction cycle (I) of the overall electron transport system of photosynthesis has been isolated [s. scheme (1) and (2)].