Isolation of intermediate compounds between hemoglobin and carbon monoxide

Area of Science:

• Biochemistry
• Protein Chemistry

Background:

• Human hemoglobin exists in various forms, including oxy-, deoxy-, carboxy-, and methemoglobin.
• Understanding the stability and interconversion of these forms is crucial for studying hemoglobin function and dysfunction.

Purpose of the Study:

• To investigate the oxidation of human hemoglobin (Hb) in a partially carbon monoxide-saturated solution.
• To characterize the formation of hemoglobin valence hybrids under specific experimental conditions.

Main Methods:

• Rapid quenching of Hb solution into a ferricyanide buffer at -25°C.
• Analysis of resulting solutions using isoelectric focusing at -25°C.
• Characterization of hemoglobin valence hybrids.

Main Results:

• Deoxyhemoglobin (deoxy Hb) rapidly oxidized to methemoglobin (met Hb), while carbon monoxide-bound hemoglobin (CO-Hb) remained largely unoxidized.
• Isoelectric focusing identified nine possible Hb valence hybrids.
• Mixing CO-Hb and methemoglobin showed minimal hybrid formation (<6%), while mixing CO-Hb and deoxyhemoglobin at 20°C led to significant hybrid formation (~10%).

Conclusions:

• Deoxy Hb is more susceptible to oxidation than CO-Hb under the studied conditions.
• The formation of Hb valence hybrids is dependent on the relative proportions of different Hb species and temperature.
• Isoelectric focusing is a valuable technique for separating and identifying Hb valence hybrids.