Characterization of Phenoloxidase Activity From Spider <i>Polybetes pythagoricus</i> Hemocyanin

Abstract

Hemocyanin of the spider <i>Polybetes pythagoricus</i>, 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 K_m and V_max of <i>Polybetes pythagoricus</i> hemocyanin (<i>Pp</i>Hc) PO activity to be 0.407mM and 0.081 μmolmin⁻¹mg protein⁻¹, respectively. Dopamine was used as the substrate with SDS at a final concentration of 10mM 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 <i>Pp</i>Hc 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 <i>Pp</i>Hc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.