Hemoglobins from Scapharca subcrenata (Bivalvia: Arcidae) likely play an bactericidal role through their peroxidase activity

Hemoglobin (Hb) is an iron-containing respiratory protein present in all vertebrates and some invertebrates. The blood clam Scapharca subcrenata is one of the few invertebrates that have Hb-containing red hemocytes. In this study, we purified Hb (Ss-Hb), including Ss-HbI and Ss-HbII, from S. subcrenata hemocytes using gel chromatography with a recovery rate of 70.71%, and then characterized their peroxidase activities. Both Ss-Hbs possessed peroxidase activity with high affinity to the substrates guaiacol and H2O2. Moreover, both Ss-Hbs had structural similarities, such as type b heme, proximal histidine (His), distal His, and heme pocket arginine (Arg), with other peroxidases. The optimal peroxidase activity of both Ss-Hbs was at pH 5 and 35 degrees C, but this was inhibited in the presence of Cu2+ and Fe2+. Ss-Hbs produced O-2(center dot-) in the presence of H2O2. beta-phenylethylamine, a substrate of peroxidase, increased the O-2(center dot-) generation, while Cu2+, an inhibitor of peroxidase, inhibited this reaction. These results indicated that the peroxidase cycle of Ss-Hb was involved in the production of O-2(center dot-). A large amount of O(2)(center dot- )may be generated by the peroxidase cycle if the substrate is sufficient. During the incubation of Ss-Hbs with Bacillus subtilis, it was speculated that trace H2O2, probably from autoxidation of Ss-Hbs or generated by B. subtilis, started the peroxidase cycle of Ss-Hb. and produced a large amount of O(2)(center dot- )in the presence of sufficient substrate in the culture medium. It is therefore reasonable to assume that Ss-Hbs played an antibacterial role owing to their peroxidase activity, which produced O-2(center dot-).