title: Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: alkaline oligomeric dissociation.
abstract: 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.
authors.affiliation: Instituto de Química de São Carlos-Universidade de São Paulo, SP, Brazil.
authors.forename: Francisco Adriano O
authors.initials: FA
authors.lastname: Carvalho
authors.forename: Patrícia S
authors.initials: PS
authors.lastname: Santiago
authors.forename: Júlio C
authors.initials: JC
authors.lastname: Borges
authors.forename: Marcel
authors.initials: M
authors.lastname: Tabak
mesh_terms: D000468:Alkalies / Q000494:pharmacology*; D000818:Animals; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D009835:Oligochaeta / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000737:chemistry; D020836:Protein Structure, Quaternary; D012984:Software; D014461:Ultracentrifugation
chemical_list: D000468:Alkalies; D006454:Hemoglobins; D010108:Oxyhemoglobins; D008623:Mercaptoethanol