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Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime

D S Gottfried1, L J Juszczak, N A Fataliev

  • 1Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. gottfrie@aecom.yu.edu

The Journal of Biological Chemistry
|January 17, 1997
PubMed
Summary

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A novel fluorescent probe, 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), quantifies binding to carboxyhemoglobin A (HbACO). This method reveals HbACO

Area of Science:

  • Biochemistry
  • Biophysics
  • Analytical Chemistry

Background:

  • Hemoglobin (Hb) allosteric regulation is crucial for oxygen transport.
  • Understanding ligand binding to Hb is key to deciphering its function.
  • Carboxyhemoglobin A (HbACO) serves as a model for studying Hb allosteric properties.

Purpose of the Study:

  • To develop and validate a time-resolved fluorescence method for quantifying ligand binding to HbACO.
  • To determine the binding site and affinity of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT) to HbACO.
  • To investigate the allosteric state of HbACO at low pH using HPT binding.

Main Methods:

  • Time-resolved fluorescence spectroscopy was employed to measure HPT fluorescence lifetimes.
  • Quantitative analysis of fluorescence decay curves determined HPT binding affinity.

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  • Competitive binding assays with inositol hexaphosphate were performed.

    • Main Results:

    • HPT exhibits distinct fluorescence lifetimes when free (5.6 ns) and bound to HbACO (~27 ps).
    • HPT binds to a single site within the central cavity of HbACO, displacing inositol hexaphosphate at a 1:1 ratio.
    • The binding site for HPT corresponds to the 2,3-diphosphoglycerate (2,3-DPG) binding site.
    • Low pH HbACO appears to exist in an altered R state, not a mixture of R and T states.

    Conclusions:

    • A novel quantitative fluorescence assay was established for measuring ligand binding to HbACO.
    • HPT serves as a reliable probe for the 2,3-DPG binding site in HbACO.
    • The findings suggest a distinct allosteric conformation of HbACO at acidic pH.