Characterization of Phenoloxidase Activity From Spider Polybetes pythagoricus Hemocyanin

LAINO, A.; LAVARIAS, S; SUAREZ, G.; LINO A.

JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY

WILEY-LISS, DIV JOHN WILEY & SONS INC

Lugar: New York; Año: 2015 vol. 323 p. 547 - 555

1932-5223

Hemocyanin of the spider Polybetes pythagoricus, in addition to its typical role as an oxygentransporter, also exhibits a phenoloxidase activity induced by micellar concentrations of SDS. Inthe present work, we found the kinetic parameters Km and Vmax of Polybetes pythagoricushemocyanin (PpHc) PO activity to be 0.407mM and 0.081 mmolmin1mg protein1, respectively.Dopamine was used as the substrate with SDS at a final concentration of 10mM and a 30-minincubation at 25°C. Conformational changes in Hc associated with the SDS treatment wereanalyzed using far-UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. Thesecondary and tertiary structural changes of PpHc induced by SDS led to increases in a-helicalcontent and tryptophan fluorescence intensity. A reduction in the absorption spectrum at 340 nmin the presence of SDS was also observed. These results suggest that the SDS-induced PO activity ofPpHc can be ascribed to conformational changes in the local environment of the typer-3 copperactive site