Phenoloxidase activity and thermostability of cancer pagurus and limulus polyphemus hemocyaninPhenoloxidase activity and thermostability of cancer pagurus and limulus polyphemus hemocyanin
Faculty of Sciences. Chemistry
Department of Chemistry
Comparative biochemistry and physiology : B : biochemistry and molecular biology. - London
164(2013):3, p. 201-209
University of Antwerp
The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, L-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and L-Dopa (K-m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for L-Dopa), while k(cat) is highest for catechol (2.44 min(-1) versus 0.67 min(-1) for L-Dopa and 0.71 min(-1) for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with L-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T-m similar to 80 degrees C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T-m similar to 92 degrees C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins. (c) 2013 Elsevier Inc. All rights reserved.