Yasuhisa Kono

Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase

Abstract Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.

Degradation of chlorophyll by nitrogen dioxide generated from nitrite by the peroxidase reaction

Abstract Nitrite, but not nitrate, added to a mixture containing horseradish peroxidase (HRP) and H 2 O 2 , bleached chlorophyll (Chl). The optimum pH for Chl bleaching was 4.0. Ascorbate, p -hydroxyphenyl acetate (HPAA), glycyltyrosine (Gly-Tyr) and amines such as morpholine and diethylamine inhibited Chi bleaching. The reaction products from HPAA and Gly-Tyr showed an absorption peak at 438 nm in an alkaline solution. The laser-Raman spectrum of the product from HPAA showed a band at 1336 cm −1 identical to that of authentic 2-nitrotyrosine. These results indicated the formation of nitrogen dioxide, the one-electron oxidation product of nitrite, which caused Chl bleaching in the nitrite/H 2 O 2 /HRP system. Nitrite caused neither inhibition of the ascorbate peroxidase reaction nor Chl bleaching with the H 2 O 2 /ascorbate peroxidase system.

Reduction of plastocyanin with O2- and superoxide dismutase-dependent oxidation of plastocyanin by H2O2.

Type I copper proteins, plastocyanin and rice blue protein, were reduced with O2 (-). The reduction rate constants of plastocyanins from several sources with O2 (-) are about 10(6) m(-1) sec(-1) (1.0 × 10(6) m(-1) sec(-1) for spinach and kidney bean plastocyanins and 6.7 × 10(5) m(-1)sec(-1) for Japanese radish plastocyanin) at pH 7.8 at 25°C and not significantly different from that observed for rice blue protein (7.3 × 10(5) m(-1)sec(-1)). Reduced plastocyanin was oxidized by H2O2 through the peroxidase-like reaction of Cu,Zn-superoxide dismutase.

Alterations in superoxide dismutase and catalase in Fusarium oxysporum during starvation-induced differentiation

Vegetative hyphae of Fusarium oxysporum differentiate into chlamydospore by triggering with carbon-starvation. The current changes in the cellular detoxifying defenses against superoxide and hydrogen peroxide: superoxide dismutase (SOD) and catalase, were examined. Although there was a little change in catalase, a dramatic change in SOD was observed during the differentiation. In vegetative hyphae of F. oxysporum f. sp. raphani, three isozymes of SOD, all of which were not inhibited by hydrogen peroxide and cyanide, were present whereas in chlamydospore an isoenzyme, which was inhibited by hydrogen peroxide but not by cyanide, was present. Thus, as differentiation proceeded, Mn-type SOD disappeared and an Fe-type SOD appeared. The results suggest that the Fe-type SOD is specifically expressed during chlamydospore formation and that active intermediates of oxygen and/or its scavenging enzymes participate in the differentiation of Fusarium oxysporum.

Cu(II)-dependent inactivation of Mn-catalase by hydroxylamine

Hydroxylamine is a strong inhibitor of the Mn-catalase of Lactobacillus plantarum in the presence of hydrogen peroxide [Kono, Y., and Fridovich, I. (1983) J. Biol. Chem. 258, 13646-13648]. In the presence of CuCl2 the Mn-catalase was rapidly inactivated by hydroxylamine without the addition of hydrogen peroxide. FeSO4 and MnCl2 were approximately 10% and 4% as effective as was CuCl2. Under anaerobic conditions, the inactivation did not occur. The chelating agents such as EDTA and histidine completely prevent the inactivation. These results indicate that the hydrogen peroxide produced during the autooxidation of hydroxylamine catalyzed by CuCl2 participates in the CuCl2-dependent inactivation by hydroxylamine.