Time-resolved fluorescence of hemoglobin species

Area of Science:

• Biophysics
• Biochemistry
• Physical Chemistry

Background:

• Hemoglobin exists in tetrameric, dimeric, and monomeric forms.
• Understanding subunit distribution is crucial for hemoglobin function.
• Previous methods lacked sensitivity to detect all forms under various conditions.

Purpose of the Study:

• To monitor hemoglobin subunit distributions (tetramers, dimers, monomers) in carbonmonoxyhemoglobin (COHb) solutions.
• To investigate how these distributions are affected by protein concentration, salt titration, and hyperbaric pressure.
• To identify molecular species in hemoglobin solutions using fluorescence lifetime measurements.

Main Methods:

• Time-resolved fluorescence spectroscopy (picosecond to nanosecond time range).
• Comparison of experimentally measured fluorescence lifetimes with computed lifetimes from atomic coordinates.
• Analysis of hemoglobin solutions under varying protein concentrations, NaCl concentrations, and hyperbaric pressures.

Main Results:

• Fluorescence lifetime analysis successfully identified tetramers, dimers, monomers, and species with disordered hemes.
• Low protein concentrations increased dimer and monomer formation.
• High NaCl concentrations promoted dimer dissociation but did not affect heme inversion or monomer formation.
• Hyperbaric pressure led to increased dimer and monomer amounts.
• Monomeric hemoglobin subunits were detected at neutral pH, a novel finding.

Conclusions:

• Time-resolved fluorescence is a powerful tool for characterizing hemoglobin quaternary structure.
• Protein concentration, ionic strength, and pressure significantly influence hemoglobin subunit equilibria.
• The detection of monomeric hemoglobin at neutral pH provides new insights into hemoglobin dynamics and stability.