Hemoglogin (Hb)
- Tetrametric, two alpha chains and two beta chains
- binds a total of 4 oxygen molecules
- carries O2 from lungs to tissues
- cooperative binding of O2
- required to increase the solubility of O2 in blood
Myoglobin (Mb)
- Monomeric
- Binds 1 oxygen molecule.
- Carries O2 from capillaries to sites of usage (mitrochondria) in cells.
- Non-cooperative binding of O2.
Properties of heme group
- Example of a prosthetic group
- Heterocylic ring containing 4 pyrrole rings
- Central atom is Fe2+ (usual oxidation state)
- Fe3+ in cytrochromes
- Mg2+ in chlorophyll
- Proximal histidine is important in transducing the binding event to protein.
- O2 binding induces a change in the electronic state of Fe2+ that changes its absorbance spectrum.
Mechanism of Positive Cooperativity in Hemoglobin:
- Binding of O2 to Fe moves proximal His residue and its attached helix (F)
- Helix F adjusts conformation by movement of a b subunits (hinge and helix ratchet)
- Alters conformation of Fe at unliganded sites.
Allosteric Effects and Cooperativity:
Allosteric effects occur when the binding properties of a macromolecule change as a consequence of a second ligand binding to the macromolecule and altering its affinity towards the first, or primary, ligand. There need not be a direct connection between the two ligands (i.e. they may bind to opposite sides of the protein)
- If the two ligands are the same (e.g. oxygen) then this is called a homotropic allosteric effect.
- If the two ligands are different (e.g. oxygen and BPG), then this is called a heterotropic allosteric effect.
In the case of macromolecules that have multiple ligand binding sites (e.g. Hb), allosteric effects can generate cooperative behavior. Allosteric effects are important in the regulation of enzymatic reactions. Both allosteric activators (which enhance activity) and allosteric inhibitors (which reduce activity) are utilized to control enzyme reactions.
Allosteric effects require the presence of two forms of the macromolecule. One form, usually called the T or tense state, binds the primary ligand (e.g. oxygen) with low affinity. The other form, usually called the R or relaxed state, binds ligand with high affinity. The T and R states are in equilibrium with each other. In the case of positive cooperativity the fraction of T states exceeds that of the R state.
Models of Allosteric Changes & Cooperativity in Hemoglobin:
Change from T to R states may occur:
- In unison (MWC model)
- Sequentially (Koshland model)
Either of these models fits the experimental data well and neither of these models are correct in describing the T ->R transition of subunits in hemoglobin.
Heterotropic Allosteric Effectors in Hemoglobin.
- BPG binds preferentially to the tense (lower binding form) of hemoglobin (see molecular models).
- It shifts the binding curve to the right, higher concentrations of oxygen are required to fully saturate hemoglobin.
- Oxygen delivery at low oxygen pressure (high altitude) is enhanced by increasing the amount of diphosphoglycerate in the red cell.
Examples of Data Analysis.
When the binding of a ligand involves a transition between a low affinity form (T) and a high affinity form (R), the two dissociation constants combine to produce a sigmoid saturation curve. The Hill equation and the Hill plot are used to determine the two Kd's and a measure of the cooperativity, the Hill coefficient, nH.
Examples of Hill plots will be shown with a description of how the slopes and intercepts can be used to extract the values of the Kd's and nH.The class hand-out, Cooperative Ligand Binding Measurements, is shown on a separate page.
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