Kinetic trapping of a key hemoglobin intermediate

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Human hemoglobin (Hb) binding involves a complex cascade of partially ligated intermediates.
• Individual intermediate binding constants are difficult to resolve directly from O(2) binding curves.
• Understanding these constants is crucial for elucidating Hb's cooperative oxygen binding mechanism.

Purpose of the Study:

• To characterize the O(2) binding constants of human hemoglobin intermediates.
• To determine the asymmetric nature of the Hb binding cascade.
• To validate a novel stopped-flow method for measuring intermediate dissociation constants.

Main Methods:

• Utilized O(2)-induced changes in the assembly constant of the α(2)β(2) tetramer from αβ dimers.
• Developed a stopped-flow approach to measure the dissociation constant of a key doubly ligated intermediate.
• Trapped released free dimers using the plasma protein haptoglobin to monitor tetramer dissociation.

Main Results:

• Characterized O(2) binding constants revealing an asymmetric Hb binding cascade.
• Demonstrated binding dependence on the specific distribution of O(2) across the four hemesites.
• Kinetic measurements confirmed previously determined equilibrium O(2) binding constants for a key intermediate.

Conclusions:

• The human hemoglobin binding cascade is inherently asymmetric.
• O(2) distribution among hemesites dictates binding affinity.
• The described stopped-flow method accurately quantifies intermediate binding constants, confirming equilibrium data.