CHARACTERIZATION OF PHENOLOXIDASE ACTIVITY FROM SPIDER POLYBETES PYTHAGORICUS HEMOCYANIN

LAINO, ALDANA; LAVARIAS, SABRIA MARIA LUISA; SUAREZ, GUSTAVO; LINO, AGUSTINA; CUNNINGHAM, MÓNICA

Journal of Experimental Zoology Part A: Ecological Genetics and Physiology

JOHN WILEY & SONS LTD

Lugar: Austin, Texas; Año: 2015 vol. 323 p. 547 - 555

1932-5231

Hemocyanin of the spider Polybetes pythagoricus shows its typical role as oxygen transporter, and also a phenoloxidase activity induced by micellar concentrations of SDS is now observed. In the present work we calculated the kinetic parameters Km and Vmax of PO activity of PpHc, which were 0.407mM and of 0.081 µmol.min-1.mg protein-1 respectively. Dopamine was used as substrate at a final concentration of 10 mM SDS and a 30-min incubation time at 25°C. Conformational changes of Hc associated with the SDS treatment were analyzed using far UV circular dichroism, intrinsic fluorescence and absorption spectroscopy. 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 340nm in the presence of SDS was observed also. These results suggest that SDS-induced PO of PpHc can be ascribed to conformational changes in the local environment of the typer-3 copper active site.