Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH

Area of Science:

• Biochemistry
• Physical Chemistry
• Protein Dynamics

Background:

• Human carboxyhemoglobin exists as a tetramer.
• Understanding protein quaternary structure transitions is crucial for biological function.

Purpose of the Study:

• To investigate the dissociation of human carboxyhemoglobin tetramers into dimers at alkaline pH.
• To quantify subunit dissociation equilibrium constants.
• To determine the charge changes associated with tetramer-dimer transition.

Main Methods:

• Photon correlation spectroscopy for diffusion studies.
• Electrophoretic light scattering for mobility measurements.
• Analysis of light scattering data to infer structural and charge properties.

Main Results:

• Confirms tetramer to dimer dissociation of carboxyhemoglobin above pH 10.
• Provides new estimates for dissociation equilibrium constants in the alkaline range.
• Electrophoretic mobilities of tetramers and dimers are similar within experimental error.
• Dissociation implies an increase of 2.8 to 4.4 net negative charges on the dimer.

Conclusions:

• Human carboxyhemoglobin undergoes tetramer-dimer dissociation at high pH.
• The dissociation is accompanied by a substantial increase in negative charge on the dimer.
• Proposed mechanisms explain the charge accumulation during dissociation.