CO binding and valency exchange in asymmetric Hb hybrids

Area of Science:

• Biochemistry
• Protein Chemistry
• Hemoglobin Research

Background:

• Controversy exists regarding the properties of asymmetric hemoglobin hybrids (unliganded dimer/liganded dimer tetramers).
• Previous dimer-tetramer equilibrium studies suggested T-like properties for these hybrids, contrasting with other findings.

Purpose of the Study:

• To investigate the conformational state of asymmetric hemoglobin hybrids using CO binding kinetics.
• To resolve conflicting experimental evidence regarding the properties of these hemoglobin species.

Main Methods:

• Photodissociation of CO ligands from [dimer-CO/dimer-azido-met] hybrids to observe CO rebinding kinetics.
• Stopped-flow apparatus to equilibrate asymmetric hybrids and measure CO binding kinetics after a delay.
• Confirmation of valency exchange in mixed hemoglobin solutions.

Main Results:

• CO rebinding kinetics to asymmetric hybrids were predominantly rapid, characteristic of the R-state (liganded tetramer).
• These rapid kinetics contradict predictions of a T-like conformation for asymmetric hemoglobin hybrids.
• Valency exchange was confirmed in mixed hemoglobin solutions, potentially explaining discrepancies in prior equilibrium studies.

Conclusions:

• Asymmetric hemoglobin hybrids display R-like kinetics, suggesting a conformation closer to the liganded state.
• The findings challenge the assignment of T-like properties based on previous dimer-tetramer equilibrium studies.
• Potential instability of asymmetric hybrids during long incubation periods, possibly due to valency exchange, may explain prior conflicting results.