Glossoscolex paulistus hemoglobin with fluorescein isothiocyanate: Steady-state and time-resolved fluorescence
| Autor: | Marina Berardi Barioni, Amando Siuiti Ito, Ana E. B. Barros, Francisco A.O. Carvalho, Marcel Tabak |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Protein Denaturation 02 engineering and technology Photochemistry Biochemistry Dissociation (chemistry) Fluorescence 03 medical and health sciences chemistry.chemical_compound Hemoglobins Structural Biology Animals Urea Oligochaeta Fluorescein isothiocyanate Molecular Biology Tryptophan General Medicine Nanosecond 021001 nanoscience & nanotechnology 030104 developmental biology chemistry HEMOGLOBINAS Hemoglobin Time-resolved spectroscopy 0210 nano-technology Fluorescein-5-isothiocyanate |
| Zdroj: | Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 1879-0003 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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