Characterization of phenoloxidase activity from spider Polybetes pythagoricus hemocyanin

Hemocyanin of the spider Polybetes pythagoricus, in addition to its typical role as an oxygen transporter, also exhibits a phenoloxidase activity induced by micellar concentrations of SDS. In the present work, we found the kinetic parameters Km and Vmax of Polybetes pythagoricus hemocyanin (PpHc) PO activity to be 0.407 mM and 0.081 µmolmin(-1) mg protein(-1) , respectively. Dopamine was used as the substrate with SDS at a final concentration of 10 mM and a 30-min incubation at 25°C. Conformational changes in Hc associated with the SDS treatment were analyzed using far-UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. The secondary and tertiary structural changes of PpHc induced by SDS led to increases in α-helical content and tryptophan fluorescence intensity. A reduction in the absorption spectrum at 340 nm in the presence of SDS was also observed. These results suggest that the SDS-induced PO activity of PpHc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.