title: Denaturant effects on HbGp hemoglobin as monitored by 8-anilino-1-naphtalene-sulfonic acid (ANS) probe.
abstract: Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB-HbGp-ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil.
authors.forename: Ana E B
authors.initials: AE
authors.lastname: Barros
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil.
authors.forename: Francisco A O
authors.initials: FA
authors.lastname: Carvalho
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil.
authors.forename: Fernanda R
authors.initials: FR
authors.lastname: Alves
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil; Universidade do Estado de Mato Grosso, MT, Brazil.
authors.forename: José W P
authors.initials: JW
authors.lastname: Carvalho
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil. Electronic address: marcel@sc.usp.br.
authors.forename: Marcel
authors.initials: M
authors.lastname: Tabak
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D000818:Animals; D006454:Hemoglobins / Q000737:chemistry*; D057446:Hydrodynamics; D006863:Hydrogen-Ion Concentration; D009835:Oligochaeta / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011489:Protein Denaturation / Q000187:drug effects*; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D006454:Hemoglobins; D010100:Oxygen
keywords: ANS probe; Denaturant chaotropic agents; Dodecyltrimethylammonium bromide; Glossoscolex paulistus hemoglobin; Optical spectroscopies