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Isolated cell-wall suspensions from horseradish in the presence of 5×10(-4) M MnCl2 catalyze the production of hydrogen peroxide at the expense of either NADPH or NADH. This reaction is inhibited by scavengers of the superoxide free radical ion such as ascorbate or dihydroxyphenols or by superoxide dismutase, and stimulated by monophenols such as p-coumaric acid. On comparison with isolated (commercial) horseradish peroxidase it becomes evident that (a) cell-wall-bound peroxidase(s) is (are) responsible for the production of hydrogenperoxide, involving the superoxide free radical ion as an intermediate of the complex reaction chain.

Formation of Nitrite from Hydroxylamine in the presence of illuminated chloroplast lamellae is inhibited by superoxide dismutase but not by catalase, indicating that the superoxide free radical ion and not H2O2 is responsible for the oxidation of hydroxylamine. Decarboxylation of α-keto acids on the other hand is strongly inhibited by catalase but only slightly by superoxide dismutase. Light-dependent hydroxylamine oxidation and decarboxylation of α-keto acids can be used, therefor, as specific and sensitive probes for the determination of either the superoxide free radical ion or hydrogen peroxide, respectively. Photosynthetic oxygen reduction in the presence of ferredoxin, (monitored by the above method) yields both H2O2 and O2·-. The addition of an oxygen reducing factor (ORF, solubilized by heat - treatment of washed chloroplast lamellae) instead of ferredoxin, however, stimulates only the production of H2O2 , while O2·- - formation is not observed. The cooperation of ferredoxin and ORF during photosynthetic oxygen reduction by chloroplast lamellae apparently produces H2O2 not only by dismutation of O2·-, but also by a separate mechanism involving ORF.

Photooxidation of hydroxylamine to nitrite by spinach (Spinacia oleracea L. and sugarbeet (Beta vulgaris L.) chloroplast lamellae in the presence of autoxidable electron acceptors is inhibited by either solubilized or membrane-bound superoxide dismutase (SOD). This inhibition is reversed by KCN. The rates of hydroxylamine photooxidation by chloroplast lamellae, a reaction which is apparently driven by the superoxide free-radical ion, was used for quantitating the amount of SOD bound to chloroplast lamellae, as compared to a soluble enzyme of defined concentration. After digitonin fragmentation of chloroplast lamellae, ca. 80% of the SOD activity is associated with subchloroplast particles sedimenting after 2 h centrifugation at 200 000 x g. Less than 10% of the SOD activity is associated with particles sedimenting after centrifugation for 30 min at 20 000 x g. 5-10% of the cyanide-sensitive SOD is recovered in the soluble fraction of the subchloroplast-free supernatant after centrifugation at 200 000 x g for 2 h.

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