title: Glossoscolex paulistus hemoglobin with fluorescein isothiocyanate: Steady-state and time-resolved fluorescence.
abstract: Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been followed, in the presence of urea, using fluorescein isothiocyanate (FITC). Binding of FITC to HbGp results in a significant quenching of probe fluorescence. Tryptophan emission decays present four characteristic lifetimes: two in the sub-nanosecond/picosecond, and two in the nanosecond time ranges. Tryptophan decays for pure HbGp and HbGp-FITC systems are similar. In the absence of denaturant, and up to 2.5mol/L of urea, the shorter lifetimes predominate. At 3.5 and 6.0mol/L of urea, the longer lifetimes increase significantly their contribution. Urea-induced unfolding process is characterized by protein oligomeric dissociation and denaturation of dissociated subunits. FITC emission decays for FITC-HbGp system are also multi-exponential with three lifetimes: two in the sub-nanosecond and one in the nanosecond range with a value similar to free probe in buffer. Increase of urea concentration leads to increase of the longer lifetime contribution, implying the removal of the quenching observed for the native HbGp-FITC system. Anisotropy decays are characterized by two rotational correlation times associated to re-orientational motions of the probe relative to protein. Our results suggest that FITC bound to HbGp is useful to monitor denaturant effects on the protein.
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
authors.forename: Ana E B
authors.initials: AE
authors.lastname: Barros
authors.affiliation: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, SP, Brazil.
authors.forename: Marina B
authors.initials: MB
authors.lastname: Barioni
authors.affiliation: Universidade Federal do Sul e Sudeste do Pará, PA, Brazil.
authors.forename: Francisco A O
authors.initials: FA
authors.lastname: Carvalho
authors.affiliation: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, SP, Brazil.
authors.forename: Amando Siuiti
authors.initials: AS
authors.lastname: Ito
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil. Electronic address: marcel@sc.usp.br.
authors.forename: Marcel
authors.initials: M
authors.lastname: Tabak
mesh_terms: D000818:Animals; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005453:Fluorescence; D006454:Hemoglobins / Q000737:chemistry*; D009835:Oligochaeta / Q000737:chemistry; D011489:Protein Denaturation; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D014364:Tryptophan; D014508:Urea; D016650:Fluorescein-5-isothiocyanate
keywords: Fluorescein isothiocyanate (FITC); Glossoscolex paulistus hemoglobin; Time-resolved fluorescence