INVESTIGADORES
LAINO Aldana
congresos y reuniones científicas
Título:
Characterization of phenoloxidase activity from spider Polybetes pythagoricus hemocyanin
Autor/es:
CUNNINGHAM MÓNICA; LAINO ALDANA; SUAREZ GUSTAVO; LINO AGUSTINA; LAVARÍAS SABRINA
Reunión:
Congreso; XII PABMB Congress; 2013
Resumen:
Hemocyanin (Hc) is generally accepted as an oxygen
transporter, although this pigment is functionally converted
into a phenoloxidase-like enzyme (PO) by so me 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 pythagorícus. Out of the three lipoproteic
fractions isolated by hemolymph ultracentrifugation of P.
pythagorícus, 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, were0.407 mmol.l-1 and 0.081 f.lmol.min-l .mg
protein-\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 develóp 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.