Francisco Adriano O. Carvalho

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.

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: analytical ultracentrifugation reexamination.

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)(3)L(3) (Vinogradov model) plus 144 heme groups, a value of M for HbGp oligomer of 3560 kDa can be predicted. This value is nearly 500 kDa higher than the unique HbGp M value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s(0)(20,w) values of 58.1+/-0.2S and 59.6+/-0.2S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600+/-100 and 3700+/-100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V(bar), for HbGp was performed based on density measurements, providing a value of 0.764+/-0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model.

Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: Alkaline oligomeric dissociation

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass (M) of 3600±100 kDa and a standard sedimentation coefficient (s20,w0) of 58 S, estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a, b, c and d plus the linker chains. The pure monomeric fraction, subunit d, and HbGp at pH 10.0, in the presence of β-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d, besides the monomeric species with s20,w0 of 2.0 S, formation of dimer of subunit d is observed with s20,w0 of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a, b, c and d, were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS.

On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea

The stability of the Glossoscolex paulistus hemoglobin (HbGp), in two iron oxidation states (and three forms), as monitored by optical absorption, fluorescence emission and circular dichroism (CD) spectroscopies, in the presence of the chaotropic agent urea, is studied. HbGp oligomeric dissociation, denaturation and iron oxidation are observed. CD data show that the cyanomet-HbGp is more stable than the oxy-form. Oxy- and cyanomet-HbGp show good fits on the basis of a two state model with critical urea concentrations at 220-222 nm of 5.1±0.2 and 6.1±0.1 mol/L, respectively. The three-state model was able to reveal a subtle second transition at lower urea concentration (1.0-2.0 mol/L) associated to partial oligomeric dissociation. The intermediate state for oxy- and cyanomet-HbGp is very similar to the native state. For met-HbGp, a different equilibrium, in the presence of urea, is observed. A sharp transition at 1.95±0.05 mol/L of denaturant is observed, associated to oligomeric dissociation and hemichrome formation. In this case, analysis by a three-state model reveals the great similarity between the intermediate and the unfolded states. Analysis of spectroscopic data, by two-state and three-state models, reveals consistency of obtained thermodynamic parameters for HbGp urea denaturation.

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.

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.

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.

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.

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.

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.