Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. II. Holo form.

The effects of guanidinium hydrochloride (GuHCl) on the functional and structural properties of a 75-kDa, functionally active hemocyanin (Hc) subunit isolated from the crab Carcinus aestuarii (holo-CaeSS2) were investigated. The holo form of the protein contains two copper ions in the active site and is capable of reversibly binding dioxygen. The present results are compared with those previously described for the corresponding functionally inactive subunit (apo-CaeSS2), devoid of the two active site copper ions (accompanying paper [R. Favilla, M. Goldoni, A. Mazzini, M. Beltramini, P. Di Muro, B. Salvato, paper published in this issue]). As with apo-CaeSS2, both equilibrium and kinetic unfolding measurements were carried out using light scattering (LS), circular dichroism, intrinsic and extrinsic fluorescence (IF and EF, respectively). In addition here, absorbance spectroscopy was exploited to evaluate oxygen binding by holo-CaeSS2. These data were combined with those relative to the protein copper content to obtain information on the stability of the active site as a function of denaturant concentration. The different techniques used revealed several unfolding transitions. At GuHCl<1 M, an appreciable increase of LS intensity was observed, about an order of magnitude lower than with apo-CaeSS2, suggesting some reversible protein aggregation. A first cooperative transition as a function of GuHCl was detected as an increase of intensity of the protein IF (C(1/2)=1 M), followed by a second transition, characterised by a small intensity decrease and a red shift of the emission maximum (C(1/2)=1.4 M). Cooperative transitions with C(1/2) values near 1.4 M GuHCl were also detected by following the decrement of: (a) EF intensity of anilino-1-naphtalenesulphonate (ANS) bound to the protein; (b) absorbance at 340 nm, typical of oxy holo-CaeSS2; (c) copper-to-protein stoichiometry. A transition at higher GuHCl (C(1/2)=1.8 M) was also observed by far UV circular dichroism (far UV CD) and related to global unfolding. Unfolding kinetics was also studied using the fluorescence stopped-flow technique. All traces were best fitted by a sum of three or four exponential terms, depending on GuHCl concentration. A comprehensive unfolding model is proposed, which accounts for most of the complex behaviour of this protein subunit, including oxy and deoxy native and aggregation-prone intermediates, a highly fluorescent intermediate, molten globule-like apo and unfolded species.