title: Isoelectric point determination for Glossoscolex paulistus extracellular hemoglobin: oligomeric stability in acidic pH and relevance to protein-surfactant interactions.
abstract: 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.
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Patrícia S
authors.initials: PS
authors.lastname: Santiago
authors.forename: Francisco Adriano O
authors.initials: FA
authors.lastname: Carvalho
authors.forename: Marco M
authors.initials: MM
authors.lastname: Domingues
authors.forename: José Wilson P
authors.initials: JW
authors.lastname: Carvalho
authors.forename: Nuno C
authors.initials: NC
authors.lastname: Santos
authors.forename: Marcel
authors.initials: M
authors.lastname: Tabak
mesh_terms: D000818:Animals; D001802:Blood Substitutes / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009835:Oligochaeta; D055503:Protein Multimerization; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D011506:Proteins; D013501:Surface-Active Agents