Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

Abstract

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0 mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0 mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204 kDa for the dodecamer (abcd)(3), 5.5S and 69 kDa for the tetramer (abcd), 4.1S and 52 kDa for the trimer (abc) and 2.0 S and 17 kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0 mol/L of denaturant, for oxy-HbGp, and above 6.0 mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0 mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations.

Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability

“…4). Recent studies of characterization of HbGp subunits by SEC and SDS-PAGE suggest that this peak corresponds to the same band of the pure native HbGp [9]. The elution volumes of the other species, as well as, previous analytical ultracentrifugation and MALDI-TOF-MS studies [29,46], imply that the peaks II, III and IV are due to the contributions of the dodecamer (abcd) 3 , trimer abc and monomer d, respectively (see Scheme 2 of the HbGp oligomeric structure and its subunits).…”

“…The increase of polydispersity index in this temperature range between 40 and 47°C indicates the existence of and equilibrium of different HbGp subunits in the solution. Previous studies on the stability of HbGp under denaturing conditions, such as, alkaline pH, have shown that the hemoglobin undergoes oligomeric dissociation into smaller species, such as, dodecamer (abcd) 3 , tetramer abcd, trimer abc and monomer d [9]. These species are characterized by hydrodynamic diameters (D H ) values of 5.1 nm for the monomer d, 7-12 nm for the mixture of trimer abc and a linker subunit, and 10-14 nm for the dodecamer (abcd) 3 [9,46].…”

“…Previous studies on the stability of HbGp under denaturing conditions, such as, alkaline pH, have shown that the hemoglobin undergoes oligomeric dissociation into smaller species, such as, dodecamer (abcd) 3 , tetramer abcd, trimer abc and monomer d [9]. These species are characterized by hydrodynamic diameters (D H ) values of 5.1 nm for the monomer d, 7-12 nm for the mixture of trimer abc and a linker subunit, and 10-14 nm for the dodecamer (abcd) 3 [9,46]. Thus, the Z-average values of 19.2 ± 0.3 and 16.3 ± 0.2 nm for the HbGp samples, in the absence and in the presence of EDTA, respectively, studied in the present work, indicate that HbGp is partially dissociated in the solution, and larger species are present in the equilibrium.…”

“…They are characterized by a very high molecular mass (MM) around 3500 kDa [2,3], a high resistance to auto-oxidation [4] and a high oligomeric stability, when exposed to different conditions of stress, such as, high temperature [5,6], pH variation [7], and addition of chemical agents [8][9][10]. The extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 MDa [11], and its oligomeric structure is composed by 144 globin chains, and 36 additional chains lacking the heme group, named linkers [12,13].…”

Estudo da estabilidade térmica da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp): efeitos do estado de oxidação do ferro do grupo heme, pH e presença de surfactante

“…15B). (Fig.15F), e o perfil da curva é consistente com a presença de subunidades dissociada em solução [19,33,58,59]. …”