Hemoglobin: Structure and Function

Question 1

1. Describe the overall structure of hemoglobin, indicating the site of oxygen binding. Explain the concepts of allostery and positive cooperativity as they relate to hemoglobin function and explain what is meant by taut (T) and relaxed (R) configurations.

Answer 1

• Hgb is a tetramer comprised of 2 alpha(-like) and 2 beta(-like) chains.
• HbA is most common in adults, HbF (alpha2gamma2) more common in fetus and newborn. Must have alpha chains to survive.
• Heme prosthetic group = protoporphyrin ring bound to iron, and iron binds to O2.
• Can only bind O2 in Fe2+ state,
• in Fe3+ state = methemoglobin.
• Reduced by cytochrome b5 reductase.
• Hgb must deliver O2 from lungs (where it easily picks it up) to tissues (where affinity decreases so it lets it go) –> aided by allostery.
• When deoxygenated, Hgb in T (taut) conformation because of its salt bonds, hydrogen bonding, and hydrophobic interactions.
• When bound to O2, bonds break and Hgb assumes R (relaxed) state.
• Allostery = substrate binding on one site –> altered conformation at other sites on protein that make it easier to bind (salt bonds break).
• makes it easier for other heme groups to bind O2 because affinity is increased –> positive cooperativity.

This allows for a sigmoid binding curve.

Question 2

2. Draw a typical oxygen dissociation curve. Explain why it is sigmoidal in shape. Define the p50. Explain the effects of pH, [CO2], temperature, and [2,3-BPG].

Answer 2

-Typical O2 dissociation curve for Hgb is sigmoid shaped (more hyperbolic for myoglobin because it is a monomer that doesn’t undergo allostery or cooperativity).
-This illustrates that Hgb binds O2 easily at high O2 concentrations in the lungs (high affinity) and lets go easily in low O2 concentrations at the tissues.
mmHg x % O2 saturation.

• P50 = partial pressure of O2 when protein is 50% saturated.
• Normal for Hgb = 27mmHg (~3 for myoglobin –> myoglobin holds O2 tightly so its better for storage).
• This is why Hgb is so good for O2 transport. “30-60, 60-90, 40-75.”
• If you increase O2 affinity and get a lower P50, curve shifts to the left (hold O2 tightly, not enough getting to the tissues).
• caused by increased pH, decreased DPG, and decreased temp.
• If you decrease O2 affinity and get a higher P50, curve shifts right (lets go O2 more easily).
• caused by decreased pH, increased DPG, and increased temp.
• pH works via the Bohr effect. From a pH 6-8.5, O2 affinity varies. O2 affinity increases in alkaline (increased pH) environments (curve shifts left), and decreases in acidic situations (decreased pH) (curve shifts right).
• Makes sense why curve shifts right during exercise, because you’re producing more bicarbonate + protons = pH drops and you get a greater release of O2 to tissues.
• Increased CO2 in blood = decreased pH = decreased O2 affinity and cure shifts right, more O2 release.

-Temperature is inversely related to oxygen affinity. High temperatures, O2 affinity decreases and more O2 released to tissues. (Exercise again.)

2,3-BPG present in red cells, product of anaerobic glycolysis.

• Binds the pocket between beta chains, stabilizes Hgb in T state = decreased O2 affinity = right shifted curve and more O2 for tissues.
• Levels increase during glycolysis, hypoxia, and anemia.

Question 3

3. Compare oxygen dissociation curves for myoglobin and hemoglobin and explain the reason for the differences.

Answer 3

Myoglobin = hyperbolic; Hgb = sigmoid.

• This is because Hgb undergoes allostery and cooperativity –> it can shift its affinity for O2 as necessary to transport oxygen.
• Once O2 binds myoglobin, the protein holds on very tightly (because it has such high affinity) until very little O2 is present (like when muscles very strained).
• This is why it’s better for storage.
• Also myoglobin is a monomer.

Question 4

4. List and describe the typical hemoglobin variants seen during fetal development and in adulthood and explain how amounts of these different hemoglobins change during development.

Answer 4

C16: zeta-alpha2-alpha1 = 2 alpha-globin genes/parent = 4 alpha genes

C11: epsilon, gammaG, gamma, delta, beta = 1 beta-globin gene/parent = 2 beta genes (so more serious if you’re missing one of those)

Embryonic = Gower 1 (zeta2epsilon2),
Gower 2 (alpha2epsilon2), and
Portland (zeta2gamma2).
-Each have higher affinity for O2 than HbA (can steal from mom)

• Fetal = HbF (alpha2gamma2, dominates after 8 weeks of gestation) (also has higher O2 affinity than HbA).
• HbF doesn’t bind BPG well so it gets stabilized in the R state and shifts curve left.
• Bohr effect helps because H+ ions transferred to maternal circulation and fetal blood pH rises = increased O2 affinity.

-Adult = HbA (alpha2beta2) and HbA2 (alpha2delta2). A2 is more heat stable and high slightly higher O2 affinity –> elevated in beta-thalassemia, HbS, hyperthyroidism, megaloblastic anemia.

More HbF after 8 gestational weeks, HbA dominates after birth because beta is more expressed, and HbF decreases because delta not expressed.
-Alpha expressed throughout so alpha-thalassemias are severe.

Question 5

5. Describe how structural differences in hemoglobin affect oxygen affinity and explain the physiologic effects of altered oxygen affinity.

Answer 5

Most common Hgb variants = HbS, HbC, HbE. –> Unstable Hgb’s, Hgb’s with altered O2 affinity, and Hgb’s associated with crystals.

• Hgb’s with altered O2 affinity usually stable with abnormal electrophoresis; no hemolysis.
• Unstable hemoglobins can denature and disrupt stability of heme-globin linkage.
• May not be detected till later, can result in altered O2 affinity, and cause hemolytic anemia (with jaundice and splenomegaly).
• Present as Heinz bodies in anemia. Generally don’t need transfusions, just folic acid. Don’t fix with splenectomy!
• Hb Koln –> common, mutation of beta chain. Increased O2 affinity and left shift.
• Hb Poole –> mutation in gamma chain = infants w/ hemolytic anemia (resolves in few months)

High-affinity Hgb’s

• Hb Chesapeake = high RBC count due to alpha-globin chain mutation = increased O2 affinity and less delivery to the tissues –> increased EPO released to stimulate erythropoiesis
• in high affinity, curve shifts left, EPO released, generally doesn’t require treatment.

Low-affinity Hgb’s

• less common; P50 right shifted because O2 released more easily
• associated with mild anemia and cyanosis
• cyanosis due to too much deoxyHb, metHb, or sulfhemoglobinemia
• inadequate oxygenation of Hgb, metHb, acquired (drugs, toxins)

Question 6

6. Describe what methemoglobinemia is, what causes it, how to diagnose it, and how to treat it.

Answer 6

• In methemoglobin, Fe is in the ferric 3+ form, which cannot carry oxygen.
• Curve shifts left, P50 goes down. Can happen due to too much metHb production (normally 1%) or because of decreased Fe reduction via cytochrome b5 reductase.
• Can also happen via oxidation of heme via reaction with free radicals.
• Different drugs can also cause.
• Can be acquired or genetic (auto recessive cytochrome b5 reductase deficiency –> increased O2 affinity, blue but well, 40% metHb, asymptomatic).
• Hemoglobin M can also be auto dominant with a mutation in alpha or beta chain that makes Fe3+ resistant to reduction. Asymptomatic cyanosis, decreased O2 affinity and Bohr effect, and increased 2,3-BPG.
• Blood presents as chocolate brown and doesn’t change when exposed to air.
• No treatment for HbM.
• Can treat methemoglobinemia with cytochrome b5 reductase to get rid of blue tinge.
• Acquired can be treated by removing the responsible drug.

Question 7

7. Explain the pathophysiology of carbon monoxide poisoning and its treatment.

Answer 7

• CO has 240x the affinity for heme than oxygen, so when it binds, it binds tightly.
• Also when it binds, it causes an allosteric change such that the other 3 heme groups don’t bind O2 as well, and curve shifts left.
• Increased changes if smoker.
• Generally presents as headache (maybe malaise, nausea, seizures, coma, and MI).
• Cherry red as opposed to cyanotic.
• 40% may walk away with neurologic deficits that stay.
• Treat with 100% O2 or hyperbaric chamber.

Question 8

8. Explain in basic terms how a pulse oximeter works. Describe situations where a pulse oximeter reading may inaccurately reflect a patient’s true oxygenation status.

Answer 8

• Pulse ox probe is a photo detector with 2 light emitting diodes.
• DeoxyHb absorbs most at 660 nm.
• Oxyhemoglobin absorbs at 940 nm.
• Carboxyhemoglobin at 660, so can give a false reading.
• Methemoglobin absorbs 660 and 940 = inaccurate.
• Co-oximetry measures at 4+ wavelengths = more accurate.
• Only measures arterial blood.
• Ideal O2 saturation between 90-100%, but elevations can change things.
• Readings can change based on probe placement, movement, nail polish, dark skin, shock, anemia, etc.