Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.Haemocyanins are oligomeric copper-bearing proteins which transport and store oxygen in a great number of arthropods and molluscs. The basic unit for arthropod haemocyanin is the hexamer, with a subunit mass of around 75 kDa. Mollusc haemocyanins are cylindrical with a subunit mass of around 400 kDa. Haemocyanins have been extensively studied as they make excellent models for investigating the multiple linkages upon which the functional behaviour of proteins is based (for a review see [l]).From a structural point of view, extensive spectroscopic studies have been carried out to elucidate the main features of the binuclear, copper-oxygen binding site. The structure of the haemocyanin of the arthropod Panulirus interruptus has been resolved by X-ray to a resolution of 0.32 nm [2] and it appears that all arthropod haemocyanins share a common architecture, according to which the basic subunit is folded into three distinct alligned domains, with the active site in the middle one.With the structural knowledge available, it seemed worthwhile exploring the energetic aspects of the folding and assembly of the molecule. Thus, we report here on the results of differential scanning calorimetry of the thermal denaturation of P . vulgaris haemocyanin in both the hexameric and monomeric forms. Differential scanning calorimetry has been widely used to study folding-unfolding processes in proteins (for reviews see [3, 41). For complex proteins that undergo reversible denaturation, the deconvolution of the experimental heat capacity profile leads to the thermodynamic characterization of those intermediate states that are significantly populated in the unfolding process, and to the definition of the structural blocks of the protein that undergo unfolding in a more or less independent fashion (see for instance . In this case, however, we have found the differential scanning calorimetry traces corresponding to the thermal denaturation of P. vulgaris haemocyanin to be irreversible and strongly scan-rate-dependent. Therefore, the analysis of these calorimetric traces has been carried out using a recently proposed approach [11, 121 which takes into account the possible kinetic character of the irreversible thermal denaturation of proteins.Correspondence to A. Parody-Morreale, Departamento de Quimica-Fisica, Facultad de Ciencias, E-18071 Granada, Spain MATERIALS AND METHODS Extraction and purification of proteinHaemocyanin was purified from lobsters (Palin...