Title: Irreversible thermal denaturation of beta-hemocyanin of Helix pomatia and its substructures studied by differential scanning calorimetry
Authors: Idakieva, K ×
Gielens, Constant
Siddiqui, NI
Doumanova, L
Vasseva, B
Kostov, G
Shnyrov, VL #
Issue Date: 2007
Publisher: Verlag der Zeitschrift für Naturforschung
Series Title: Zeitschrift für Naturforschung. A, A journal of physical sciences vol:62 issue:9 pages:499-506
Abstract: The thermal denaturation of beta-hemocyanin from the gastropod Helix pomatia (beta-HpH) at neutral pH was studied by means of differential scanning calorimetry (DSC). The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning previously scanned samples. Two transitions, with apparent transition temperatures (T-m) of ca. 84 degrees C (main transition) and ca. 88 degrees C (minor transition), were detected by DSC in 20 mm MOPS buffer, containing 0.1 m NaCl, 5 mm CaCl2 and 5 mm MgCl2 at pH 7.2 (buffer A), using a heating rate of 1.0 K min(-1). Both T-m values were dependent on the scanning rate, suggesting that the thermal denaturation of beta-HpH is a kinetically controlled process. The T-m and specific enthalpy values (Delta H-cal) for the thermal denaturation of beta-HpH were found to be independent of the protein concentration, indicating that the dissociation of the protein into monomers does not take place before the rate-determining step of the process of thermal unfolding started. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. These transitions are tentatively attributed to the denaturation of, respectively, the wall (main transition) and the collar of the beta-HpH molecule. The activation energies (E-A) of both transitions were found to be similar (about 500 kj mol(-1)).
In 130 mm glycine/NaOH buffer, pH 9.6 (buffer B), with beta-HpH dissociated into subunits, the calorimetric profile had a more complex character. This could be ascribed to a different stability of the functional units (FUs) constituting the beta-HpH subunit. FU d, which in the cylindrical didecameric beta-HpH molecule is located in the wall, was markedly less stable than FU g, which belongs to the collar. The thermal denaturation of FUs d and g was described by the two-state irreversible model. On the basis of this model, the parameters of the Arrhenius equation were calculated.
ISSN: 0932-0784
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

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