Oligomeric stability studies of giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the presence of chaotropic agents, surfactants and characterization of its subunits

Glossoscolex paulistus hemoglobin (HbGp) is characterized by a molecular mass of 3.6 MDa, a high oligomeric stability, a high resistance to oxidation and a high affinity to oxygen. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains lacking the heme group, named linkers, organized in a double-layered hexagonal structure. In this current work the characterization of the HbGp subunits and the effect of pH and urea upon the oligomeric stability were studied by several biophysical techniques. Our results obtained by electrophoresis SDS-PAGE, MALDI-TOF-MS and analytical ultracentrifugation (AUC) showed that only the monomer d isolated by size exclusion chromatography (SEC) presented high purity. For the other fractions various species were observed in the solution. Thus, for the trimeric fraction, two species are present in the equilibrium, the main species with percentage contribution of 87 % is assigned to the trimer abc and the species with 13 % in the solution is associated to the complex (abc + L). Additionally, the data obtained by several spectroscopic techniques and AUC show clearly that the oligomeric stability of HbGp depends on the iron oxidation state, the specific ligand coordinated to the iron and the protein concentration. Therefore, our results show that the met-HbGp form is the less stable one in the alkaline medium and in the presence of urea, followed by the oxy- and cyanomet- forms. In this way, at pH 8.0, the met- form is fully dissociated into smaller subunits, such as, trimer abc and monomer d, while the oxy-HbGp is partially dissociated with a significant percentage contribution (88 %) of undissociated protein, and the cyanomet-HbGp does not undergo oligomeric dissociation. The sedimentation coefficients (s20,w) and molecular masses (MM) values for species present in the solution, at different pH, are very close to the values obtained for isolated species. In the presence of urea the same behavior was observed for the three HbGp forms as compared to the alkaline medium. However, for a full characterization of the unfolding process the thermodynamic parameters were obtained by spectroscopic data analysis using models of two and three states. Adequate fits were obtained for both models, but the three states model was very appropriate to describe the HbGp denaturation process. Thus, the denaturation process of HbGp is defined by two phases. The first phase between 1.0 and 3.0 mol/L, of urea is assigned to the transition of native state to an intermediate state (N → I), and is characterized by dissociation of the oligomer in several subunits. The strong similarity of the intermediate state to the native one suggests that oligomeric dissociation induces little changes in the secondary structure and the region of heme group of the protein. As a consequence, the thermodynamic parameters associated to the first transition have large errors due to the complexity of the intermediate state with different species in the solution, as well as its great similarity to the native state. The second phase (I → U), associated with a cooperative transition at 4,5 - 5,0 mol/L of denaturant agent, is attributed to the unfolding of the dissociated subunits. Our AUC and SAXS data are very consistent with spectroscopic data. Thus, in the first phase the oligomeric dissociation of whole protein in dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d was observed. For urea concentrations above 4.0 - 5,0 mol/L, for oxy-HbGp and cyanomet-HbGp, respectively, the significant increase in I(0), Dmax and Rg values suggests that the HbGp subunits are denatured in the solution. The molecular masses values (MM) obtained by mass spectrometry and AUC, and the sedimentation coefficients (s20,w) are consistent with others results reported in the literature for orthologous hemoglobins. In addition, the results of this work correspond to an important advance in the characterization of the denaturation process of this complex oligomeric protein. (AU)