A sensitive assay based upon fluorescence of scopoletin allowed continuous recording of H20, production in illuminated intact cells of Anacytis nidulans. Onset of illumination was followed by a 5 to 10 second lag, a burst of very rapid production continuing for up to 5 minutes, and finally a slow and continuing steady rate of H202 production. Size of the H202 burst was decreased by 3-(3,4-dichlorophenyl)-1,l-dimethvlurea, by low 02, and by certain Calvin cycle intermediates; it was increased by high light intensity, CO2 depletion, Calvin cycle inhibitors (as iodoacetamide), cold shock, carbonyl cyanide ,n-chlorophenylhydrazone, and certain organic acids as glycolate). The H202 burst was explained by the following hypothesis: a low potential reductant is produced more rapidly than it can be used in the normal pathway to C02 reduction and, instead, reacts with oxygen. H20. production is regarded as a metabolic defect observable in Anacystis most dramatically during the transition from a very low rate of oxidative dark nmetabolism to a high rate of photosynthetic metabolism.The classical experiments of demonstrated the production of H202 by isolated chloroplasts. In subcellular preparations the role of molecular oxygen as a Hill oxidant and production of H202 has been well established, even in the absence of autoxidizable additives (3,7,15,17,20). However, it has remained uncertain whether reduction of oxygen to H202 is a normal event or an artifact induced by damage to the photosynthetic apparatus during isolation. Very small amounts of H202 production have been reported in flashilluminated cells of Chlorella (l1) and in Anacystis nidulatns (38). We now report the direct, continuous measurement of photosynthetic production of H.O by intact cells of Anacystis nidulans.
MATERIALS AND METHODSModification of the scopoletin fluorescence assay (. 34) allowed continuous recording of production of H202 by illuminated algal cells. Scopoletin is oxidized by H202 in the presence of peroxidase so that decrease in scopoletin fluorescence is a direct measure of amount of H202. The assay apparatus is described in Figure 1. The routine assay reaction mixture con-'This study was supported by Grant GM 11300 from the National Institutes of Health.2Present address: Division of Biological Sciences, Indiana University, Bloomington, Ind. 47401. tained 1 nmole of scopoletin and 80 units of peroxidase in a total volume of 3.3 ml. Cuvette temperature did not rise significantly above room temperature (24-26 C). We confirmed the previously reported requirement for peroxidase and the 1:1 stoichiometry for scopoletin-H20,2 in our apparatus using H202 solutions standardized against KMnO,. Oxygen exchange was measured by a Beckman oxygen macroelectrode in a cuvette of about 1.5 ml at a sensitivity of about 0.02 ,l 02/ml (32).Anacystis nidulans Richter (strain Tx 20 of our laboratory) was grown in medium C (24) in a continuous culture apparatus at 25 C or 30 C, aerated with 1% CO2 in air. Although no detailed study of culture conditions ...
