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A Photochemically Active Particle Derived from Chloroplasts by the Action of the Detergent Triton X-100

From the ¹ From the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387

Treatment of spinach chloroplasts with 4% Triton X-100 yields fragments which are separable by centrifugation. In the medium used (which is 4% in Triton X-100 and 0.5 m in sucrose) for the initial incubation particles sedimented by centrifugation at 144,000 x g for 1, 3, and 8 hours are relatively inactive in the partial photoreactions representative of photosynthesis. These particles contain an increased amount of chlorophyll b when compared with chloroplasts. These particles could represent Pigment System 2 which is involved in oxygen evolution in the intact chloroplast but is inactivated by the detergent treatment.

The supernatant fluid from the centrifugation described above contains particles which are sedimented by 10 hours of centrifugation at 144,000 x g after diluting with an equal volume of water. These particles exhibit a high rate of nicotinamide adenine dinucleotide phosphate photoreduction, with rates as high as 1980 µmoles of NADP photoreduced per hr per mg of chlorophyll observed. This reaction requires the presence of spinach ferredoxin, ferredoxin-NADP reductase, and plastocyanin in addition to an electron donor system of ascorbate-2,6-dichloroindophenol (ascorbate alone is only moderately active) or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. This activity is heat-labile, being destroyed by heating to 40° for 5 min. Other photochemical reactions supported by this particle include methyl red reduction (supported by ascorbate-dichloroindophenol), ferrocytochrome c photooxidation (aerobic), and ferricytochrome c photoreduction (supported by reduced trimethylbenzoquinone). Methyl red photoreduction activity is also destroyed by heating, while ferricytochrome c photoreduction activity is only partially destroyed by heating to 70°. Methyl red photoreduction and ferrocytochrome c photooxidation also are stimulated by plastocyanin.

The photoactive particles contain low amounts of chlorophyll b, show an unresolved cytochrome spectrum indicating the presence of cytochrome f and b₆, exhibit an increased absorbance at 700 mµ, and show a greatly enhanced P700 absorption change and electron spin resonance spectrum upon illumination. The particle is only weakly fluorescent and does not emit any delayed light following illumination. All these properties indicate that this particle retains the photoactive Pigment System 1 which is responsible for NADP photoreduction in photosynthesis.

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