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

• HbGp is an oligomeric extracellular hemoglobin, with molecular mass of 3.6 MDa.
• CTAC induces a partial dissociation of oxy-HbGp 0.5 mg/mL, at pH 5.0, 20 °C.
• At pH 9.0, oxy-HbGp dissociation is complete at 30 °C, in the presence of CTAC.
• The oxy-HbGp dissociation is a necessary step, in the thermal denaturation process.
• Dissociation, unfolding and aggregation processes depend on the pH and HbGp concentration.

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 3.6 MDa. 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 0.5 mg/mL, undergoes a partial oligomeric dissociation, on going from 0.2 to 0.6 mmol/L of CTAC, accompanied by a decrease in the Dh values from 27 ± 1 to 22 ± 1 nm. 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 0.2 mmol/L of CTAC, while in the presence of 0.4 mmol/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.

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