Hemoglobin

Question 1

What are the basic characteristics of hemoglobin and myoglobin?

Answer 1

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

Question 2

Picture of Heme subunit.

Answer 2

Question 3

Second picture of heme subunit.

Answer 3

Question 4

What does the heme subunit consit of?

Answer 4

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⁺

Question 5

How does O₂ bind to hemoglobin?

Answer 5

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

Question 6

What is cooperativity?

Answer 6

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.

Question 7

Describe the conformational change that occurs when oxygen binds to iron in the heme.

Answer 7

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)

Question 8

How does the quaternary structure change communication between subunits influence the change in the heme molecule?

Answer 8

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 15^o 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

Question 9

What does the T and R state of hemoglobin refer to?

Answer 9

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

Question 10

Picture of the concerted model of oxygen binding.

Answer 10

T state is deoxyhemoglobin

R state is high affinity for oxygen hemoglobin

Question 11

Picture of sequential model of hemoglobin binding to oxygen.

Answer 11

Question 12

2nd picture of sequential binding of hemoglobin to oxygen.

Answer 12

Question 13

What is allosteric regulation?

Answer 13

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.

Question 14

What is 2,3-Bisphosphoglycerate?

Answer 14

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.

Question 15

Picture showing impact of 2,3-BPG, pH, and Temperature on oxgen delivery to tissues.

Answer 15

Question 16

How does 2,3-Bisphosphoglycerate decrease hemoglobins O² affinity?

Answer 16

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.

Question 17

Describe the binding of 2,3 BPG to hemoglobin.

Answer 17

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.

Question 18

How does 2,3 BPG affect the oxygen dissociation curve?

Answer 18

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

Question 19

How does 2,3 BPG respond to chronic hypoxia or anemia?

Answer 19

The concentration of 2,3 BPG will increase during conditions of chronic hypoxia or anemia.

Question 20

How is 2,3 BPG related to fetal hemoglobin (HbF)?

Answer 20

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

Question 21

What is the composition of fetal hemoglobin?

Answer 21

HbF is composed of two alpha-like zeta chains and two beta-like gamma chains.

Question 22

How does low pH impact hemoglobins affinity for O² binding and what affect does this have on the oxygen dissociation curve?

Answer 22

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

Question 23

How does CO₂ binding to hemoglobin impact its oxygen binding affinity?

Answer 23

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

Question 24

Graph of factors that influence oxygen dissociation curve.

Answer 24

Temperature

pH

2,3-BPG

CO

CO₂

pH

Question 25

What is the form of hemoglobin that is predominate in sickle cell disease?

Answer 25

Hemoglobin F (HbF)

Question 26

What is the composition / make up of HbS?

Answer 26

HbS contains two normal alpha globins and two mutant beta globins

Question 27

What amino acid substitution in the HbS beta chains is responsible for the abnormal hemoglobin?

Answer 27

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

Question 28

Describe the concerted model of hemoglobin binding.

Answer 28

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).

Question 29

How does pH and CO₂ affect oxygen release from hemoglobin?

Answer 29

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

Question 30

What are the sources of protons that lower pH?

Answer 30

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

Question 31

Which form of hemoglobin has a higher affinity for protons?

Answer 31

Deoxyhemoglobin has a higher affinity for protons. This is due to specific histidine side chains that have a higher pK_a in the deoxyhemoglobin state

Question 32

How does 2,3-bisphosphoglycerate impact oxygen affinity in hemoglobin?

Question 33

Describe the binding of 2,3 bpg to hemoglobin.

Answer 33

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

Question 34

How does 2,3 BPG respond to chronic hypoxia and anemia?

Answer 34

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

Question 35

What is the relationship of fetal hemoglobin (HbF) and 2,3-BPG?

Answer 35

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.

Question 36

Describe O₂ binding to hemoglobin.

Answer 36

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

Question 37

Describe allosteric effectors of deoxyhemoglobin.

Answer 37

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

Question 38

What does binding to carbon monoxide lead to where hemoglobin is concerned?

Answer 38

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

Question 39

Describe the pathology of HbS.

Answer 39

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