Hemoglobin Flashcards
What are the basic characteristics of hemoglobin and myoglobin?
Hemoglobin and myoglobin are both O2 transport and storage proteins
Myoglobin is specific for muscles
Myoglobin is a monomeric protein; Hemoglobin is tetrameric
Myoglobin is made up of 153 amino acids and has a molecular weight of 17,200
Hemogobin is made up of two alpha subunits with 141 amino acid residues and two beta subunits made up of 146 amino acid residues
Picture of Heme subunit.
Second picture of heme subunit.
What does the heme subunit consit of?
Fe2+ is incorporated into the prosthetic group called heme
A prosthetic group is a compound associated with a protein that is necessary for the proteins function
Heme consists of a protoporphyrin which has a singe Fe2+ bound.
The iron has four bounds to nitrogen atoms that are part of the porphyrin ring and two bonds to that are perpendicular to the porphyrin ring
Tne nitrogen atoms which are nucleophilic help prevent the Fe2+ from converting to Fe3+
How does O2 bind to hemoglobin?
When oxygen first binds to Fe2+ in the heme of hemoglobin, the iron is drawn into the plane of the porphyrin ring
This causes a series of conformational changes that are transmitted to adjacent subunits
As the iron atom moves into the plane of the heme, histidine F8 and its associated residues are pulled along with the iron atom. This overcomes steric hinderance.
This in turn increases the adjacent subunits affinity for oxygen
This is an example of positive cooperativity
What is cooperativity?
Cooperative binding occurs if the number of binding sites of a macromolecule that are occupied by a specific type of ligand is a non-linear function of this ligand’s concentration. This can be due, for instance, to an affinity for the ligand that depends on the amount of ligand bound.
Describe the conformational change that occurs when oxygen binds to iron in the heme.
When oxygen is not bound, Fe2+ is out of the heme plane
Oxygen binding pulls the Fe2+ into the heme plane
This pulls Histidine F8 wlong with it because the F helix moves when oxygen binds the Fe2+
Total movement of the Fe2+ is 0.029 nm (0.29 A)
How does the quaternary structure change communication between subunits influence the change in the heme molecule?
The carboxy terminal end of the F helix lies in the interface between the two alpha and beta subunits
When Fe2+ is pulled into the plane of the heme plane, salt bridges are broken between the C terminal regions
One alpha-beta subunit pair moves relative to the other by 15o upon oxygen binding
The change in position of the carboxy terminal ends of the helix favors the T to R transition
This change is induced by Fe binding to oxygen
This structural transition at the iron ion in one subunit is directy transmitted to the other subunits
What does the T and R state of hemoglobin refer to?
The T and R states of hemoglobin are structural descriptions of the molecule.
The T and R states are always at equilibrium with one another
In the T state, the porphyrin rings are dome-shaped and has a lower affinity for O2
In the R state, the porphyrin rings are flat and planar and the heme molecule has a higher affinity for oxygen
In the T state, the binding of protons (a low pH) stabilizes and decreases its O2 affinity
Picture of the concerted model of oxygen binding.
T state is deoxyhemoglobin
R state is high affinity for oxygen hemoglobin
Picture of sequential model of hemoglobin binding to oxygen.
2nd picture of sequential binding of hemoglobin to oxygen.
What is allosteric regulation?
Allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein’s allosteric site (that is, a site other than the protein’s active site).
Effectors that enhance the protein’s activity are referred to as allosteric activators, whereas those that decrease the protein’s activity are called allosteric inhibitors.
What is 2,3-Bisphosphoglycerate?
2,3-BPG is a cmpound that is formed from 1,3-BPG in peripheral tissues.
In the absence of 2,3-BPG, oxygen binding to Hb follows a rectangular hyperbolic curve.
The sigmoid binding curve is only seen in the presence of 2,3-BPG.
Picture showing impact of 2,3-BPG, pH, and Temperature on oxgen delivery to tissues.
How does 2,3-Bisphosphoglycerate decrease hemoglobins O2 affinity?
2,3-BPG decreases Hb O2 affinity by binding to the deoxyhemoglobin state and not the oxyhemoglobin state.
This preferential binding stabilizes the T state of deoxyhemoglobin.
Because the R state is more stable, little oxygen would be released under physiological conditions.
Describe the binding of 2,3 BPG to hemoglobin.
One (1) molecule of 2,3-BPG will bnd to a pocket formed by two beta globin clusters.
This pocket lies at the center of the deoxyhemoglobin tetramer.
The + charges on amino acids (2 lysines and 4 histidines) within the pocket form ionic bonds (salt bridges) with - charge phosphate groups on BPG.
Because 2,3 BPG binds at a site distant from where the iron molecule binds in hemoglobin, it is considered an allosteric inhibitor.
How does 2,3 BPG affect the oxygen dissociation curve?
When 2,3 BPG is removed from hemoglobin, it has a high affinity for O2
When bound with 2,3 BPG however, the affinity goes down and the dissociation curve shifts to the right
This reduced affinity allows hemoglobin to release O2 more easily
How does 2,3 BPG respond to chronic hypoxia or anemia?
The concentration of 2,3 BPG will increase during conditions of chronic hypoxia or anemia.
How is 2,3 BPG related to fetal hemoglobin (HbF)?
In HbF, the g subunit has a Ser21 instead of a His21; no salt bridge can form
This allows HbF to move from the T to R state more easily
It has a higher affinity for O2 HbA
2,3 BPG does not bind as easily to HbF as it does HbA
What is the composition of fetal hemoglobin?
HbF is composed of two alpha-like zeta chains and two beta-like gamma chains.
How does low pH impact hemoglobins affinity for O2 binding and what affect does this have on the oxygen dissociation curve?
Low pH will shift the O2 binding curve of hemoglobin downwards and to the right
At low pH, hemoglobins O2 binding affinity is decreased
Results in more oxygen being delivered to tissues
When ionic bonds break to relax T state to R state, protons are released
Oxygen binding to the R state diminishes proton binding
How does CO2 binding to hemoglobin impact its oxygen binding affinity?
CO2 binding stabilizes the T state and reduces oxygen binding affinity
It does this by reacting with the terminal amino groups to form carbamate groups
Carbamates favor the formation of salt bridges between the a and b chains that stabilize the T state
CO2 also reacts with water to form H2CO3 which dissociates into bicarbonate and hydrogen ions
This drops pH which leads to a stabilization of the T state
Graph of factors that influence oxygen dissociation curve.
Temperature
pH
2,3-BPG
CO
CO2
pH
What is the form of hemoglobin that is predominate in sickle cell disease?
Hemoglobin F (HbF)
What is the composition / make up of HbS?
HbS contains two normal alpha globins and two mutant beta globins
What amino acid substitution in the HbS beta chains is responsible for the abnormal hemoglobin?
Glutamate at position six is replaced by valine.
This makes the beta globins less negative
The replacement of the glutamate by valine causes a protrusion on the hemoglobin molecule
This protrusion fits into a complementary site on another HbS molecule and causes long strands of the molecule to accumulate
These strands cause RBC’s to “sickle” and these cells will occlude blood vessels and cause severe anoxia
Describe the concerted model of hemoglobin binding.
The concerted model of allostery, postulates that hemoglobin subunits are connected in such a way that a conformational change in one subunit is necessarily conferred to all other subunits.
Thus, all subunits must exist in the same conformation. The model further holds that, in the absence of any ligand (substrate or otherwise), the equilibrium favours one of the conformational states, T or R.
The equilibrium can be shifted to the R or T state through the binding of one ligand to a site that is different from the active site (the allosteric site).
How does pH and CO2 affect oxygen release from hemoglobin?
The release of oxygen from hemoglobin is enhanced when pH is lowered or when hemoglobin in the presence of increased CO2 conc.
Both lead to decreased oxygen affinity of hemoglobin and a right-shift on the oxygen dissociation curve and a stabilization of the T state of hemoglobin
This is known as the Bohr effect
Raising pH or lowering CO2 conc. results in greater affinity of hemoglobin for oxygen.
This leads to a left-shift on the oxygen dissociation curve and a stabilization of the R state of hemoglobin
What are the sources of protons that lower pH?
The protons come from CO2 being converted (by carbonic anhydrase) to carbonic acid
Carbonic acid spontaneously loses an H+ and becomes bicarbonate
These two source contribute to lowering pH
Which form of hemoglobin has a higher affinity for protons?
Deoxyhemoglobin has a higher affinity for protons.
This is due to specific histidine side chains that have a higher pKa in the deoxyhemoglobin state
How does 2,3-bisphosphoglycerate impact oxygen affinity in hemoglobin?
Describe the binding of 2,3 bpg to hemoglobin.
One molecule of 2,3 bpg binds to a pocket formed by the 2 beta globin chains in the center of the tetramer
This pocket contains positively charged amino acids (His143, Lys82, and His2) that form ionic bonds with 2,3 bpg (the negatively charged phosphates)
This binding shifts the oxygen dissocation curve to the right and hemoglobin releases its oxygen more efficiently
How does 2,3 BPG respond to chronic hypoxia and anemia?
In response to chronic hypoxia and anemia, the levels of 2,3-BPG increase
This lowers the afinity of of hemoglobin for oxygen and allows more efficient delivery of oxygen to the tissues
What is the relationship of fetal hemoglobin (HbF) and 2,3-BPG?
Fetal blood has a higher O2 affinity than the mother’s blood. In the fetus the chains are replaced by similar, but distinctly different chains, so fetal hemoglobin (Hbf) has an 2 2 structure.
The intrinsic oxygen affinity of Hbf is very similar to that of HbA, but Hbf has a much lower affinity for BPG than does HbA.
This difference is largely due to the replacement of His 143 in the adult chain by a serine in the fetal chain.
The positively charged His 143 in adult chains helps to bind the negatively charged BPG molecule, favoring the deoxy form.
The concentration of BPG is about the same in the circulatory systems of mother and fetus. Under these conditions, Hbf will have less BPG bound than will HbA, and therefore Hbf will have a higher oxygen affinity at the same BPG concentration.
Describe O2 binding to hemoglobin.
The first O2 that binds to the first heme subunit does so with low affinity
This starts the transition from the T state to the R state
The next three O2 that bind do so with sequentially increasing binding affinities
This leads to the sigmoid curve that is seen in hemoglobin oxygen dissociation curves
Describe allosteric effectors of deoxyhemoglobin.
Hydrogen (H+ )
2,3-Bisphosphoglycerate
CO2
All lead to stabilization of the T state and a decreased affinity for O2
This leads to increased oxygen dilevery and a right shifted dissociation curve
What does binding to carbon monoxide lead to where hemoglobin is concerned?
CO binding to hemoglobin leads to carboxyhemoglobin
It has a high affinity for CO
This stabilizes the R state
This in turn leads to a higher binding affinity for O2
This decreases oxygen delivery to the tissues and leads to a left shift of the O2 dissociation curve
The curve assumes a hyperbolic look
Describe the pathology of HbS.
HbS is caused by a point mutation in both of the beta globin genes
Glu is substituted for Val
This results in a decreased solubility of deoxyhemoglobin
This leads to polymer formation as the hemeoglobin molecules aggregate
This ultimately leads to vascular occlusion and pain
