Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin.
The intrinsic and inducible phenoloxidase (PO) activity of Rapana thomasiana hemocyanin (RtH) and its substructures were studied. With catechol as substrate, a weak o-diPO activity was measured for the didecameric RtH and its subunits. Some activation of the o-diPO activity of RtH was achieved by limited treatment with subtilisin and by incubation of RtH with 2.9 mM sodium dodecyl sulphate (SDS), suggesting an enhanced substrate access to the active sites. The highest artificial induction of o-diPO activity in RtH, however, was obtained by lyophilization of the protein. This is ascribed to conformational changes during the lyophilization process of the didecameric RtH molecules, affecting the accessibility of the active sites. These conformational changes must be very small, since Fourier-transform infrared and circular dichroism spectroscopies did not reveal any changes in secondary structure of lyophilized RtH. The difference in accessibility of the copper containing active site for substrates between catechol oxidase and functional unit RtH2-e was demonstrated by molecular modeling and surface area accessibility calculations. The low level of intrinsic PO activity in the investigated hemocyanin is related to the inaccessibility of the binuclear copper active sites to the substrates.
Idakieva
Krassimira
K
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str. Bl. 9, Sofia 1113, Bulgaria. idakieva@orgchm.bas.bg
Siddiqui
Nurul Islam
NI
Meersman
Filip
F
De Maeyer
Marc
M
Chakarska
Irena
I
Gielens
Constant
C
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005612:Freeze Drying; D006433:Hemocyanins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D029623:Primulaceae / Q000201:enzymology*; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D003902:Detergents; D012967:Sodium Dodecyl Sulfate; D006433:Hemocyanins; D014442:Monophenol Monooxygenase