title: pH effect upon HbGp oligomeric stability: characterization of the dissociated species by AUC and DLS studies.
abstract: 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.
authors: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
authors: Francisco Adriano O
authors: FA
authors: Carvalho
authors: José Wilson P
authors: JW
authors: Carvalho
authors: Fernanda Rosa
authors: FR
authors: Alves
authors: Marcel
authors: M
authors: Tabak
mesh_terms: D000818:Animals; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008970:Molecular Weight; D009835:Oligochaeta / Q000737:chemistry*; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification; D058767:Protein Unfolding; D012542:Scattering, Radiation; D014461:Ultracentrifugation; D014508:Urea / Q000737:chemistry
chemical_list: D006454:Hemoglobins; D021122:Protein Subunits; D006418:Heme; D014508:Urea; D007501:Iron