Cinnabarinate formation in malpighian tubules of the silkworm. Bombyx mori: reaction mechanism of cinnabarinate formation in the presence of catalase and manganese ions.

The formation of cinnabarinate in the presence of manganese ions and catalase was investigated spectrophotometrically. The absorption peak of cinnabarinate at 460 nm appeared only in a reaction system containing manganese ions and catalase. If catalase was omitted, a new absorption peak at 360 nm was observed while the absorption peak of cinnabarinate reached a plateau. Furthermore, in the presence of hydrogen peroxide, the absorption spectrum of cinnabarinate changed; catalase suppressed this absorption change. We conclude that in the course of cinnabarinate formation in the presence of manganese ions hydrogen peroxide is produced, which decomposes cinnabarinate. Catalase prevents the accumulation of hydrogen peroxide, which results in the steady increase of cinnabarinate. Cinnabarinate formation by manganese ions shows an initial lag phase. This lag phase disappeared by preincubating 3-hydroxyanthranilate under aerobic conditions. Incubation of 3-hydroxyanthranilate resulted in the generation of superoxide anions. When both manganese ions and superoxide anions were present, the lag phase of cinnabarinate formation disappeared. In the process of cinnabarinate formation, manganese ions serve to dismutate superoxide anions, as does superoxide dismutase; manganese (II) ions were oxidized to manganese (III) ions by superoxide anions. From these results we have proposed a mechanism of cinnabarinate formation catalysed by manganese ions.