Interaction of human apohemoglobin with inositol hexaphosphate

Area of Science:

• Biochemistry
• Protein Chemistry
• Structural Biology

Background:

• Apohemoglobin, the protein component of hemoglobin without its heme groups, can exist in various polymeric states.
• Understanding protein polymerization is crucial for comprehending protein function and stability.
• Inositol hexaphosphate (IP6) is a highly charged molecule known to interact with proteins.

Purpose of the Study:

• To investigate the effect of inositol hexaphosphate (IP6) on the polymerization state of apohemoglobin.
• To elucidate the structural and conformational changes induced by IP6 binding to apohemoglobin.
• To determine the stoichiometry of IP6 binding to apohemoglobin.

Main Methods:

• Sedimentation velocity and equilibrium ultracentrifugation to analyze apohemoglobin polymerization.
• Optical rotatory dispersion (ORD) spectroscopy to assess changes in helical content.
• Circular dichroism (CD) spectroscopy in the near-UV region to probe aromatic amino acid environments.
• Chemical modification assays using p-mercuribenzoate and N-ethylmaleimide to study reactivity of beta-93 cysteinyl residues.

Main Results:

• IP6 shifts apohemoglobin polymerization towards tetramer formation, with optimal effect at 1 mM IP6.
• Higher IP6 concentrations (above 1 mM) promote redissociation into dimers.
• IP6 binding suggests a stoichiometry potentially higher than 1 mol IP6 per 4 apohemoglobin subunits.
• ORD spectra indicate a slight increase in apohemoglobin's helical content upon IP6 interaction.
• Near-UV CD spectra show no significant alteration in the environment of aromatic residues (tyrosine, phenylalanine, tryptophan).
• IP6 decreases the reaction rate of beta-93 cysteinyl residues with sulfhydryl-reactive reagents, suggesting tertiary structure changes.

Conclusions:

• Inositol hexaphosphate modulates apohemoglobin polymerization in a concentration-dependent manner.
• IP6 induces conformational changes in apohemoglobin, affecting both secondary and tertiary structures.
• The interaction involves IP6 binding to apohemoglobin, influencing its quaternary structure and the accessibility of specific residues.