title: Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin.
abstract: 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 4mM 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)~80 °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)~92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.
authors: Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 9, Sofia 1113, Bulgaria. idakieva@orgchm.bas.bg
authors: Krassimira
authors: K
authors: Idakieva
authors: Yuliana
authors: Y
authors: Raynova
authors: Filip
authors: F
authors: Meersman
authors: Constant
authors: C
authors: Gielens
mesh_terms: D000818:Animals; D033381:Anomura / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D002396:Catechols / Q000378:metabolism; D002850:Chromatography, Gel; D004298:Dopamine / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006433:Hemocyanins / Q000378:metabolism*; D006458:Hemolymph / Q000201:enzymology; D006737:Horseshoe Crabs / Q000201:enzymology*; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002396:Catechols; D007980:Levodopa; D006433:Hemocyanins; D014442:Monophenol Monooxygenase; C034221:catechol; D004298:Dopamine