title: Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies.
abstract: Oxy-HbRa thermal stability was evaluated by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) at pH 5.0, 7.0, 8.0, and 9.0. DLS results show that oxy-HbRa, at pH 7.0 and 5.0, remains stable up to 56 °C, undergoing denaturation/aggregation in acidic media above 60 °C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 °C, before denaturation. SAXS data show that oxy-HbRa, at 20 °C, is in its native form, displaying radius of gyration (R g) and particle maximum dimension (D max) of 108 ± 1 and 300 ± 10 Å, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 °C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 °C, accompanied by an increase of R g and D max values. However, an overlap of oligomeric dissociation and denaturation in the system is observed upon temperature increase, with an increase in R g and D max. Analysis of experimental p(r) curves as a linear combination of theoretical curves obtained for HbGp fragments from the crystal structure shows an increasing contribution of dodecamer (abcd)3 and tetramer (abcd) in solution, as a function of pH values (8.0 and 9.0) and temperature. Finally, our data show, for the first time, that oxy-HbRa, in neutral and acidic media, does not undergo oligomeric dissociation before denaturation, while in alkaline media the oligomeric dissociation process is an important step in the thermal denaturation.
authors.affiliation: Universidade do Estado de Mato Grosso, Barra do Bugres, MT, Brazil. jwilsonc@unemat.br.
authors.forename: José Wilson P
authors.initials: JW
authors.lastname: Carvalho
authors.affiliation: Instituto de Química de São Carlos, SP, Brazil.
authors.forename: Francisco A O
authors.initials: FA
authors.lastname: Carvalho
authors.affiliation: Universidade Estadual Paulista Júlio de Mesquita Filho, Registro, SP, Brazil.
authors.forename: Patrícia S
authors.initials: PS
authors.lastname: Santiago
authors.affiliation: Instituto de Química de São Carlos, SP, Brazil.
authors.forename: Marcel
authors.initials: M
authors.lastname: Tabak
mesh_terms: D000818:Animals; D000067493:Dynamic Light Scattering; D005110:Extracellular Space; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009835:Oligochaeta / Q000166:cytology*; D010108:Oxyhemoglobins / Q000737:chemistry; D011489:Protein Denaturation; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D053838:Scattering, Small Angle; D013696:Temperature; D014961:X-Ray Diffraction
chemical_list: D006454:Hemoglobins; D010108:Oxyhemoglobins
keywords: Denaturation/aggregation; Erythrocruorins; Oligomeric dissociation; Thermal stability; pH