Characterization of phenoloxidase activity from spider Polybetes pythagoricus hemocyanin

CUNNINGHAM MONICA; LAINO ALDANA; LINO AGUSTINA; SUAREZ GUSTAVO; LAVARIAS SABRINA

Puerto Varas

Congreso; XLIX Annual Meeting Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2013

Hemocyanin (Hc) is generally accepted as an oxygen transporter, although this pigment is functionally converted into a phenoloxidase-like enzyme (PO) by some reagents including SDS. PO is present in almost all organisms, functioning as an initiator of melanin synthesis. The aim of this work is to characterize PO enzymatic activity in spider Polybetes pythagoricus. Out of the three lipoproteic fractions isolated by hemolymph ultracentrifugation of P. pythagoricus, VHDL was separated, which contains hemocyanin as the major apoprotein and transports most of the circulating lipids. PO activity was assayed using dopamine as substrate by spectrophotometrical method at 475 nm. After testing several incubation times with SDS at a concentration of 10 mM, PO activity developed only after 20-min incubation. The kinetics parameters, Km and Vmax, were 0.407 mmol.l-1 and 0.081 μmol.min-1.mg protein-1, respectively. Hill coefficient was approximately 1 indicating Michaelis-Menten behavior. Great temperature dependence was observed with an optimum value at 42°C. It was performed a delipidation control of VHDL by SDS-PAGE-analysis, using the same SDS concentration as in enzymatic assays. From the observed results we can conclude that Hc is a very stable protein, which needs an exhaustive treatment to develop PO activity in comparison with Hc of other arachnids. This work was supported by grants from: CONICET-PIP no. 075 and PICT no. 2010-1270, Argentina.