Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies.

Oxy-HbRa thermal stability was evaluated by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) at pH 5.0, 7.0, 8.0, and 9.0. DLS results show that oxy-HbRa, at pH 7.0 and 5.0, remains stable up to 56 °C, undergoing denaturation/aggregation in acidic media above 60 °C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 °C, before denaturation. SAXS data show that oxy-HbRa, at 20 °C, is in its native form, displaying radius of gyration (R g) and particle maximum dimension (D max) of 108 ± 1 and 300 ± 10 Å, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 °C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 °C, accompanied by an increase of R g and D max values. However, an overlap of oligomeric dissociation and denaturation in the system is observed upon temperature increase, with an increase in R g and D max. Analysis of experimental p(r) curves as a linear combination of theoretical curves obtained for HbGp fragments from the crystal structure shows an increasing contribution of dodecamer (abcd)3 and tetramer (abcd) in solution, as a function of pH values (8.0 and 9.0) and temperature. Finally, our data show, for the first time, that oxy-HbRa, in neutral and acidic media, does not undergo oligomeric dissociation before denaturation, while in alkaline media the oligomeric dissociation process is an important step in the thermal denaturation.

Interaction of cationic dodecyl-trimethyl-ammonium bromide with oxy-HbGp by isothermal titration and differential scanning calorimetric studies: Effect of proximity of isoelectric point.

In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy-form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB-oxy-HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher Tm values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy-HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy-HbGp-DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy-HbGp-cationic surfactant interaction at neutral pH.

Glossoscolex paulistus extracellular hemoglobin (HbGp) oligomeric dissociation upon interaction with sodium dodecyl sulfate: Isothermal titration calorimetry (ITC).

Annelid erythrocruorins are respiratory proteins with high cooperativity and low autoxidation rates. The giant extracellular hemoglobin of the earthworm, Glossoscolex paulistus (HbGp), has a molecular mass of 3.6 MDa. In this work, isothermal titration calorimetry (ITC), together with DLS and fluorescence emission have been used to investigate the interaction of SDS with the HbGp in the oxy-form, at pH 7.0. Our ITC and DLS results show that addition of SDS induces oxy-HbGp oligomeric dissociation, while a small amount of protein aggregation is observed only by DLS. Moreover, the oligomeric dissociation process is favored at lower protein concentrations. The temperature effect does not influence significantly the interaction of SDS with the hemoglobin, due to the similarities presented by the critical aggregation concentration (cac) and critical micelle concentration (cmc') for the mixtures. The increase of oxy-HbGp concentration leads to a slight variation of the cac values for the SDS-oxy-HbGp mixture, attributed mainly to the noncooperative electrostatic binding of surfactant to protein. However, the cmc' values increase considerably, associated to a more cooperative hydrophobic binding. Complementary pyrene fluorescence emission studies show formation of pre-micellar structures of the mixture already at lower SDS concentrations. This study opens the possibility of the evaluation of the surfactant effect on the hemoglobin stability by ITC, which is made for the first time with this extracellular hemoglobin.

Cetyltrimethylammonium chloride (CTAC) effect on the thermal stability of oxy-HbGp: dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies.

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, displaying a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6MDa. CTAC effects on the oxy-HbGp thermal stability were investigated, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS data show that the oxy-HbGp-CTAC interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0 and 7.0. In the acidic pH, oxy-HbGp 0.5mg/mL, undergoes a partial oligomeric dissociation, on going from 0.2 to 0.6mmol/L of CTAC, accompanied by a decrease in the Dh values from 27±1 to 22±1nm. It is observed, for the first time, that in the absence and in the presence of CTAC, oxy-HbGp undergoes a partial oligomeric dissociation, with increase of temperature, before denaturation and aggregation at pH values 7.0 and 5.0. SAXS data show that oxy-HbGp undergoes denaturation at 60°C, in the presence of CTAC, pH 5.0. At neutral pH 7.0, the aggregation process starts at 20°C, with increase of Rg and Dmax parameters. At both pH values, 5.0 and 7.0, the denaturation and aggregation are accompanied by the sedimentation of the aggregates. At pH 9.0, oxy-HbGp is totally dissociated at 40°C, in the presence of 0.2mmol/L of CTAC, while in the presence of 0.4mmol/L of surfactant the aggregation process starts at 20°C, with the full denaturation of protein at higher temperature. Finally, our data show, for the first time, that the oligomeric dissociation is an important step in the thermal denaturation of oxy-HbGp, in the presence of CTAC, independently of both the pH and the protein concentration.

Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies.

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, presenting a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6 MDa. SDS effects on the oxy-HbGp thermal stability were studied, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS and SAXS data show that the SDS-oxy-HbGp interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0. At pH 7.0, oxy-HbGp undergoes complete oligomeric dissociation, with increase of temperature, in the presence of SDS. Besides, oxy-HbGp 3.0mg/mL, pH 7.0, in the presence of SDS, has the oligomeric dissociation process reduced as compared to 0.5mg/mL of protein. At pH 9.0, oxy-HbGp starts to dissociate at 20 °C, and the protein is totally dissociated at 50 °C. The thermal dissociation kinetic data show that oxy-HbGp oligomeric dissociation at pH 7.0, in the presence of SDS, is strongly dependent on the protein concentration. At 0.5mg/mL of protein, the oligomeric dissociation is complete and fast at 40 and 42 °C, with kinetic constants of (2.1 ± 0.2) × 10(-4) and (5.5 ± 0.4) × 10(-4) s(-1), respectively, at 0.6 mmol/L SDS. However, at 3.0mg/mL, the oligomeric dissociation process starts at 46 °C, and only partial dissociation, accompanied by aggregates formation is observed. Moreover, our data show, for the first time, that, for 3.0mg/mL of protein, the oligomeric dissociation, denaturation and aggregation phenomena occur simultaneously, in the presence of SDS. Our present results on the surfactant-HbGp interactions and the protein thermal unfolding process correspond to a step forward in the understanding of SDS effects.

pH effect upon HbGp oligomeric stability: characterization of the dissociated species by AUC and DLS studies.

Glossoscolex paulistus (HbGp) extracellular hemoglobin is a giant oligomeric protein. It is constituted by 144 heme containing subunits and non-heme structures (linkers), with a total molecular mass of 3.6MDa. AUC and DLS studies were developed for three HbGp forms, oxy-, met- and cyanomet-, at several pH values, in order to characterize the species in solution upon oligomeric dissociation. Isolated SEC fractions, trimer and dodecamer, are less stable as compared to the whole oxy-HbGp. The monomer d displays a large thermal stability up to 59°C. Hydrodynamic properties of the isolated subunits are very similar to those described for them in the whole protein, in the presence of urea or at pH 10.0. The degree of HbGp oligomeric dissociation, in alkaline pH, depends significantly on the iron oxidation state. Also on the ligand coordinated to the heme iron. Thus, at pH 8.0, the oxy-HbGp is partially dissociated, while the met-form is fully dissociated. The cyanomet-HbGp remains undissociated. Our present results show that the effect of pH on the HbGp oligomeric stability is similar to that associated to the urea-induced unfolding. Simultaneous use of AUC and DLS allowed the characterization of the species in the SEC fractions of isolated HbGp subunits.

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

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.

Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media.

The thermal denaturation and aggregation of the HbGp, in the oxy- and cyanomet-forms, was investigated by DSC, AUC, DLS, optical absorption and CD, in the pH range from 5.0 to 7.0. Oxy-HbGp has a denaturation process partially reversible and dependent on the temperature. DSC melting curve is characterized by a single peak with T(c) value of 333.4 ± 0.2K for oxy-HbGp, while two peaks with T(c) values of 332.2 ± 0.1 and 338.4 ± 0.2K are observed for cyanomet-HbGp, at pH 7.0. In acidic pH oxy- and cyanomet-HbGp are more stable showing higher T(c) values and aggregation. AUC data show that, HbGp, at pH 7.0, upon denaturation, remains undissociated at 323 K, presenting oligomeric dissociation at 333 (12 ± 3% of tetramer and 88 ± 5% of whole HbGp) and 343 K (70 ± 5% of monomer and 30 ± 2% of trimer). DLS data show that the lag period before aggregation is dependent on the temperature and HbGp concentration. Optical absorption and CD results show that the increase of temperature leads to the oxy-HbGp oxidation and aggregation, above 331 K, in acidic pH. CD data, for HbGp, present a greater thermal stability in acid medium than at neutral pH, with similar T(c) values for both oxidation forms. Our data are consistent with previous studies and represents an advance in understanding the thermal stability of oligomeric HbGp structure.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies.

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 °C, and Rg is 108 Å, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 °C and 60 °C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 °C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of Rg, Dmax and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet->oxy->met-HbGp.

Thermal stability of extracellular hemoglobin of Glossoscolex paulistus: determination of activation parameters by optical spectroscopic and differential scanning calorimetric studies.

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS), optical absorption spectroscopy (UV-VIS) and differential scanning calorimetry (DSC). At pH 7.0, cyanomet-HbGp is very stable, no oligomeric dissociation is observed, while denaturation occurs at 56°C, 4°C higher as compared to oxy-HbGp. The oligomeric dissociation of HbGp occurs simultaneously with some protein aggregation. Kinetic studies for oxy-HbGp using UV-VIS and DLS allowed to obtain activation energy (E(a)) values of 278-262 kJ/mol (DLS) and 333 kJ/mol (UV-VIS). Complimentary DSC studies indicate that the denaturation is irreversible, giving endotherms strongly dependent upon the heating scan rates, suggesting a kinetically controlled process. Dependence on protein concentration suggests that the two components in the endotherms are due to oligomeric dissociation effect upon denaturation. Activation energies are in the range 200-560 kJ/mol. The mid-point transition temperatures were in the range 50-65 °C. Cyanomet-HbGp shows higher mid-point temperatures as well as activation energies, consistent with its higher stability. DSC data are reported for the first time for an extracellular hemoglobin.

Isoelectric point determination for Glossoscolex paulistus extracellular hemoglobin: oligomeric stability in acidic pH and relevance to protein-surfactant interactions.

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 MDa. It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole pH range. Our zeta-potential data are consistent with light scattering results. Average values of pI obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at pH 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5.0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.