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Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin
Idakieva, K.; Meersman, F.; Gielens, C. (2012). Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1824(5): 731-738. dx.doi.org/10.1016/j.bbapap.2012.03.002
In: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. Elsevier: Amsterdam. ISSN 1878-1454, more
Peer reviewed article  

Available in Authors 
    VLIZ: Open Repository 291775 [ OMA ]

Keyword
    Marine
Author keywords
    Thermal stability; Differential scanning calorimetry; FTIR spectroscopy;Hemocyanin; Mollusca

Authors  Top 
  • Idakieva, K.
  • Meersman, F., more
  • Gielens, C., more

Abstract
    Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV-vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60 degrees C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80 degrees C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50 kW functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal beta-sheet rich domain of the functional unit starts to unfold before the alpha-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90 degrees C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc. Crown Copyright

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