Glossoscolex paulistus hemoglobin with fluorescein isothiocyanate: Steady-state and time-resolved fluorescence

- Fluorescein isothiocyanate (FITC) binds to Glossocolex paulistus hemoglobin (HbGp) at pH 7.0.
- Protein/heme molar ratios of 0.2 and 2.0 lead to 0.12 and 0.79 FITC molecules bound per heme.
- Protein tryptophans emission show greater sensitivity to urea as compared to FITC emission.
- Time-resolved fluorescence decays were studied for both protein tryptophans and bound FITC.
- Fluorescence anisotropy decays show two rotational correlation times for HbGp-bound FITC.

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 2.5 mol/L of urea, the shorter lifetimes predominate. At 3.5 and 6.0 mol/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.