DYE REDUCTION BY ILLUMINATED CHLOROPLAST FRAGMENTS

It has been demonstrated that when isolated chloroplasts are illuminated in the presence of aqueous solutions of certain oxidante, oxygen evolution occurs (2, 5, 6, 7, 8,13). Provided stoichiometric relationships between the reactants and products prevail, the reaction rate can be measured in terms of oxygen production or in terms of changes in the concentration of whatever other molecular or ionic species are involved, e.g., changes in the concentration of the oxidant, of the reduction product, or of H+. Of these possibilities, oxygen production, measured by manometric or colorimetric methods, has been most commonly used. Measurements of decrease in the oxidant concentration or increase in the concentration of the corresponding reduction product appear to be limited to determinations with highly colored reagents, e.g., dyes. Hydrogen ion formation or disappearance, which allows the use of acid-base titrations, is limited to special cases, as for example, reduction of an oxidant such as ferricyanide (7). From the foregoing possible methods only two appear to be adaptable to rapid determinations of reaction rates when micro amounts of chloroplast material are to be used. These are: (a) colorimetric determination of oxygen as oxyhemoglobin by the method of Hill (5), and (b) determination of the decolorization of oxidation reduction indicators (8). Such micro methods require the elimination of secondary influences which can be neglected in manometric determinations. In the determination of oxygen as oxyhemoglobin it is necessary to remove oxygen by preliminary degassing of the reagents and chloroplast material. In the case of reduction of indophenol dyes, the presence of small quantities of natural reducing substances, such as ascorbic acid, introduces a thermal reduction which might invalidate the observations. In addition to thermal reductions not involving O2 production, photochemical reduction of certain dyes may occur, which likewise does not involve Oa production. Such reduction was found when m?thyl?ne blue or thionine was added to the supernatant fraction from a brei made from