Hemoglobin and Myoglobin

What is myoglobin?

Oxygen storage protein in heart and skeletal muscles; a monomer that contains one heme-binding site that binds one oxygen

What is hemoglobin?

Oxygen carrier protein from lungs to tissues; a tetramer that contains four heme groups and binds up to 4 oxygen molecules

Hemoglobin’s quaternary structure determines it’s…?

Oxygen-binding behavior; allows T-to-R state transitions between subunits, producing cooperative oxygen binding and efficient oxygen release in tissues

What are the characteristics of myoglobin’s oxygen-binding curve?

Has a single polypeptide chain and a hyperbolic oxygen-saturation curve; binds O2 quickly and tightly, even at low pO2, has a very low P50, indicating high O2 affinity

What are the characteristics of hemoglobin’s oxygen-binding curve?

Has four interacting subunits and a sigmoidal oxygen-saturation curve; binding of one O2 increases the affinity of the remaining subunits, positive cooperativity allows hemoglobin to load oxygen efficiently in the lungs at high pO2

What does the higher P50 of hemoglobin indicate?

Lower O2 affinity; allows hemoglobin to release oxygen in tissues at lower pO2

What is P50?

The partial pressure of oxygen when 50"% of binding sites are occupied

What is the physiologic consequence of cooperativity?

Makes hemoglobin highly responsive over the pO2 range between lungs and tissues; hemoglobin remains highly saturated in the lungs but releases a large fraction of oxygen when pO2 falls in tissues

Cooperative hemoglobin delivers about __% of its bound oxygen from lungs to tissues?

66%

Myoglobin delivers about __% if its bound O2 from lungs to tissues?

7%

Hypothetical noncooperative hemoglobin would deliver only about __% of its bound oxygen to tissues?

38%

What is heme?

The O2 binding prosthetic group in myoglobin and hemoglobin

What does the globin fold create?

A heme pocket in which the proximal histidine (His F8) coordinates iron; positioning it for reversible O2 binding

What is the site of O2 binding?

Central ferrous iron; only ferrous iron can reversibly bind and release oxygen

What are the features of histidine?

pH sensitive; low pH favors O2 unloading (Bohr effect)

What helps maintain iron in the ferrous state?

The nonpolar heme pocket limits oxidation

What does oxidation of ferrous to ferric form?

Methemoglobin, which cannot bind oxygen

What are the features of methemoglobin?

Cannot bind oxygen, makes remaining bound oxygen harder to unload

What reduces ferric back to ferrous?

NADH-cytochrome b5 reductase

What can cause methemoglobinemia?

Oxidant drugs/chemicals, enzyme deficiency, or HbM mutations

How can HbM variants occur?

Alpha or beta globin mutations, including substitution of the proximal histidine His F8 with tyrosine, which stabilizes ferric and promotes methemoglobin formation

What are the clinical features of methemoglobinemia?

Cyanosis, dark red “chocolate-covered” blood, and misleading pulse oximetry

What is the treatment for methemoglobinemia?

Methylene blue

Where does CO bind?

The heme iron of hemoglobin, forming carboxyhemoglobin

Hemoglobin has a larger affinity for what molecule?

CO

What effect does CO have on hemoglobin?

Stabilizing the R state, reducing oxygen release to tissues

What is the effect of CO on blood?

Decreases the oxygen-carrying capacity of blood

What is the treatment for CO poisoning?

Binding is reversible; treated with 100% oxygen therapy

CO binding shifts the oxygen-hemoglobin dissociation curve…?

Left; reducing oxygen release to tissues

What does pulse oximetry measure?

Estimates arterial oxygen saturation noninvasively

What is SpO2?

Percentage of hemoglobin’s oxygen-binding sites occupied by oxygen

What are the light absorptions compared in a pulse oximeter?

Red light (660nm, absorbed more by deoxy hemoglobin) and Infrared light (940nm, absorbed more by oxy hemoglobin)

What is the benefit of pulse oximetry?

It can detect hypoxemia before visible cyanosis

What are the limitations of pulse oximetry?

CO poisoning and methemoglobinemia can make SpO2 misleading because the device is calibrated for oxy and deoxy hemoglobin

What are the allosteric effects that influence O2 binding and release from hemoglobin?

Hydrogen (acid), CO2, and 2,3-bisphosphoglycerate

What is the effect of acid (low pH) on O2 binding?

Promotes O2 release in acidic tissues

What is the effect of CO2 on O2 binding?

Binds to globin, stabilizes the deoxy (T) form to release O2

What is the effect of 2,3-bisphosphoglycerate on O2 binding?

Helps release O2 in tissues, especially at high altitudes, exercise, hypoxic conditions, anemia

Is myoglobin affected by allosteric effects?

No

What is the mechanism behind low pH and oxygen binding?

A decrease in pH (increased H+) in peripheral tissues stabilizes the deoxy (T) form of hemoglobin, which alters the subunit interaction and decreases oxygen affinity of hemoglobin; this results in oxygen release at tissues and shifts oxygen saturation curve to the right

What is the Bohr effect?

The change in O2 affinity of hemoglobin in response to acid and CO2

What is the mechanism behind binding of CO2 to hemoglobin?

CO2 binds covalently to the N-terminal amino groups of deoxy hemoglobin as carbamates, which stabilizes the deoxy conformation; formation of carbamates releases H+ and contributes to the Bohr effect

Most of the CO2 produced by metabolism in tissues is transported to the lungs as…?

Bicarbonate (HCO3-) in the plasma

What favors the unloading of oxygen in peripheral tissues and loading of oxygen in the lungs?

The differential pH gradient (tissues have lower pH and lungs have higher pH)

What is the mechanism behind carbonic acid formation and O2 unloading?

1. CO2 enters the RBC

2. Carbonic anhydrase catalyzes CO2 to H2CO3

3. H2CO3 dissociates to H+ and HCO3-

4. Increased H+ protonates hemoglobin

5. Hemoglobin affinity for oxygen decreases and oxygen released to tissues

What is the mechanism behind CO2 unloading and O2 loading?

1. O2 enters the RBC

2. O2 binding to hemoglobin releases H+ and forms HbO2 (oxyhemoglobin)

3. H+ combines with HCO3- to form H2CO3

4. Carbonic anhydrase catalyzes H2CO3 to CO2 and H2O

5. CO2 diffuses into alveoli and is exhaled

What is the mechanism behind 2,3-Bisphosphoglycerate and the reduction in O2 affinity?

1. 2,3-Bisphosphoglycerate is produced in RBCs

2. 2,3-BPG binds positively charged residues (Lys and His) in the beta chains, stabilizing deoxy hemoglobin in the T state and promoting oxygen release

Increases in response to chronic hypoxia; oxygenation of hemoglobin in the lungs changes its conformation and 2,3-BPG cannot bind to oxy hemoglobin

What is the main issue of fetal blood and oxygen?

Oxygen must move from maternal blood to fetal blood; placental pO2 is relatively low, fetal hemoglobin must therefore load oxygen efficiently at lower pO2

Adult hemoglobin consists of?

2 alpha and 2 beta subunits

Fetal hemoglobin consists of?

2 alpha and 2 gamma subunits

What is the functional difference between fetal and maternal hemoglobin?

Binding of 2,3-BPG to fetal hemoglobin is weaker than that of adult hemoglobin, resulting in increased affinity of fetal hemoglobin for oxygen than maternal hemoglobin

How does fetal hemoglobin shift the oxygen binding curve?

To the left, and increases the oxygen delivery to the fetus

Where is the alpha-globin gene family found?

Chromosome 16

What are the major forms of hemoglobin in adults?

HbA (alpha2, beta2), HbA2 (alpha2, delta2), and HbA1C (alpha2, beta2, glucose)

Where is the beta-globin gene family found?

Chromosome 11

What are the chains made from the alpha hemoglobin family?

Alpha 1, Alpha 2, Zeta

What are the chains made from the beta hemoglobin family?

Epsilon, gamma, delta, and beta

What are hemoglobinopathies?

Genetic disorders caused by the production of structurally abnormal hemoglobin or insufficient quantities of hemoglobin

What are the two categories of hemoglobin mutations?

Qualitative and quantitative abnormalities

What are qualitative abnormalities of hemoglobin?

Mutations that make a normal amount of altered hemoglobin (amino acid changes); like sickle cell

What are quantitative abnormalities of hemoglobin?

Mutations that make a normal hemoglobin, but in a reduced amount of each chain; like thalassemias

What is the most common qualitative hemoglobinopathy mutation?

Sickle cell

What is the inheritance of sickle cell anemia?

Autosomal recessive

What is the mutation in sickle cell anemia?

Point mutation in beta-globin (Glutamate → Valine) E6V

What is the mechanism of sickle cell anemia?

Mutation creates a hydrophobic “sticky patch” that causes deoxy hemoglobin to polymerize into long fibers (RBC sickling); vaso-occlusion, hypoxia, and impaired blood flow occur. Rigid sickled RBCs become trapped in capillaries and are destroyed, which causes chronic hemolytic anemia

What are the features of chronic hemolysis?

Hyperbilirubinemia (jaundice), low hematocrit, and increased reticulocytes

How is sickle cell managed/treated?

Hydroxyurea can increase gamma globin synthesis and fetal hemoglobin; bone marrow transplant

What is the heterozygous advantage of sickle cell?

Protection from malaria [example of balanced polymorphism]

What is hemoglobin C?

Glutamate to lysine at position 6 of the beta globin; RBCs show decreased flexibility; comigrates with HbA2 on electrophoresis

How is electrophoresis used to diagnose hemoglobinopathies?

Hemoglobins are negatively charged and migrate toward the anode; can be used to see what hemoglobins are present

What are thalassemias?

Inherited disordered caused by reduced globin chain production

What are the clinical features of moderate to severe thalassemias?

Anemia, fatigue, jaundice, splenomegaly, abdominal swelling, and growth delay

What are characteristic of most severe beta-thalassemia?

Bone and facial changes

What are characteristic of most severe alpha-thalassemia?

HbH disease or hydrops fetalis

What is Beta Thalassemia minor?

When one beta globin allele has a mutation, individuals will have microcytosis and mild (or no) anemia

What is Beta Thalassemia major?

When both beta-globin alleles have mutations

What is Cooley Anemia?

Complete lack of beta-globin; requires transfusions

Excess alpha chain leads to…?

Alpha-chain precipitation; causes severe microcytic and hypochromic anemia

What treatment is suggested for all thalassemia patients with evidence of hymolysis?

Folate supplements

What can happen as a result of frequent blood transfusions?

Iron overload; which can treated with iron chelators that bind iron and increase its secretion

What does anemia-driven erythropoiesis cause?

Suppresses hepcidin, allowing ferroportin to increase intestinal iron absorption

How many alpha-globin genes do humans possess?

4

If ¾ alpha-globin genes are deleted, what occurs?

HbH disease (beta4 tetramers)

If all alpha-globin genes are deleted, what occurs?

Hb Bart Disease (gamma4 tetramers)

Normal hemoglobin is glycated non-enzymatically to form…?

HbA1c

Why are HbA1C levels elevated in patients with diabetes?

Persistently high glucose levels during the lifetime of RBCs