title: Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin.
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°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°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 50kDa 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 β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90°C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.
authors.affiliation: Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria. idakieva@orgchm.bas.bg
authors.forename: Krassimira
authors.initials: K
authors.lastname: Idakieva
authors.forename: Filip
authors.initials: F
authors.lastname: Meersman
authors.forename: Constant
authors.initials: C
authors.lastname: Gielens
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry*; D006433:Hemocyanins / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008974:Mollusca / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry*; D058767:Protein Unfolding; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D021122:Protein Subunits; D003300:Copper; D006433:Hemocyanins; D010100:Oxygen