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T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Published on: July 31, 2010
Ligand linked assembly of Scapharca dimeric hemoglobin
W E Royer1, R A Fox, F R Smith
1Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA. royer@darwin.ummed.edu
The Journal of Biological Chemistry
|February 28, 1997
Summary
Cooperativity in Scapharca dimeric hemoglobin is not due to quaternary enhancement. Instead, the protein
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Hemoglobin cooperativity is crucial for efficient oxygen transport.
- The assembly of hemoglobin quaternary structure influences its allosteric properties.
- Scapharca dimeric hemoglobin serves as a model for studying hemoglobin assembly and function.
Purpose of the Study:
- To investigate the role of quaternary enhancement in the cooperativity of Scapharca dimeric hemoglobin.
- To test the hypothesis that tighter dimer assembly in ligated states enhances oxygen affinity.
- To elucidate the mechanism underlying cooperativity in this dimeric hemoglobin.
Main Methods:
- Analytical gel chromatography to assess protein assembly.
- Sedimentation equilibrium experiments to determine association constants.
- Oxygen binding experiments to measure oxygen affinity and cooperativity.
Main Results:
- Dissociation of the dimer was observed in liganded states (CO and oxygenated) with association constants in the range of 10^8 M^-1.
- The deoxy dimer showed no detectable dissociation, indicating a tighter assembly in the unligated state.
- Oxygen binding experiments revealed increased oxygen affinity and decreased cooperativity at lower concentrations, refuting quaternary enhancement.
Conclusions:
- The hypothesis of quaternary enhancement is unambiguously refuted for Scapharca dimeric hemoglobin.
- The deoxy interface is more hydrated and more tightly assembled than liganded interfaces.
- Quaternary constraint, rather than quaternary enhancement, underlies cooperativity in Scapharca dimeric hemoglobin, similar to other allosteric proteins.
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