Spectroscopic properties and conformational stability of Concholepas concholepas hemocyanin.
The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.
Idakieva
Krassimira
K
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Akad. G. Bonchev-Str. Bl. 9, Sofia, Bulgaria. idakieva@orgchm.bas.bg
Nikolov
Peter
P
Chakarska
Irena
I
Genov
Nicolay
N
Shnyrov
Valery L
VL
D020106:Acrylamide / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D049851:Gastropoda / Q000737:chemistry*; D006433:Hemocyanins / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D021122:Protein Subunits / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D021122:Protein Subunits; C021724:oxy-beta-hemocyanin; D020106:Acrylamide; D006433:Hemocyanins