Guanidine hydrochloride and urea effects upon thermal stability of Glossoscolex paulistus hemoglobin (HbGp).

Glossoscolex paulistus hemoglobin (HbGp) has a molecular mass of 3600kDa. It belongs to the hexagonal bilayer hemoglobin class, which consists of highly cooperative respiratory macromolecules found in mollusks and annelids. The present work focusses on oxy-HbGp thermal stability, in the presence of urea and guanidine hydrochloride (GuHCl), monitored by several techniques. Initially, dynamic light scattering data show that the presence of GuHCl induces the protein oligomeric dissociation, followed by a significant 11-fold increase in the hydrodynamic diameter (DH) values, due to the formation of protein aggregates in solution. In contrast, urea promotes the HbGp oligomeric dissociation, followed by unfolding process at high temperatures, without aggregation. Circular dichroism data show that unfolding critical temperature (Tc) of oxy-HbGp decreases from 57°C, at 0.0 mol/L of the denaturant, to 45°C, in the presence of 3.5 mol/L of urea, suggesting the reduction of HbGp oligomeric stability. Moreover, differential scanning calorimetry results show that at lower GuHCl concentrations, some thermal stabilization of the hemoglobin is observed, whereas at higher concentrations, the reduction of stability takes place. Besides, HbGp is more stable in the presence of urea when compared with the guanidine effect, as deduced from the differences in the concentration range of denaturants.

Denaturant effects on HbGp hemoglobin as monitored by 8-anilino-1-naphtalene-sulfonic acid (ANS) probe.

Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB-HbGp-ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.

Characterization of Rhinodrilus alatus hemoglobin (HbRa) and its subunits: evidence for strong interaction with cationic surfactants DTAB and CTAC.

Rhinodrilus alatus is an annelid and its giant extracellular hemoglobin (HbRa) has a molecular mass (MM) of 3500kDa. In the current study, the characterization of MM values of the HbRa subunits, and the effects of surfactants and alkaline pH upon HbRa stability were monitored. Electrophoresis, MALDI-TOF-MS and AUC show that the MM values of HbRa subunits are very close, but not identical to the Glossoscolex paulistus hemoglobin (HbGp). The monomer d is found to exist in, at least, two isoforms: the main one, d1, displays a MM of 16,166±16Da, and the second one, d2, is less intense with MM of 16,490±20Da. For the trimer abc and tetramer abcd, single contributions around 51,470Da and 67,690Da were observed, respectively. Finally, the monomers a, b, and c, present MM values of 17,133, 17,290 and 15,506Da, respectively. Both CTAC and DTAB interact strongly with HbRa, and up to seven surfactant molecules are bound to the protein. On the other hand, spectroscopic studies show that HbRa is more stable at alkaline pH, as compared to HbGp. Thus, our data suggest that alkaline medium, up to pH10.0, induces the oligomeric dissociation, without promoting the subunits unfolding and heme iron oxidation. Our results suggest that the MM of the annelid hemoglobin subunits is conserved to a great extent in the evolution process of these species.