Proteins: Myoglobin and Hemoglobin

Why study oxygen-binding proteins?

Essential for aerobic metabolism; O2 solubility in blood is very low; specialized proteins transport and store oxygen

Overview of myoglobin

Monomeric protein; single polypeptide chain; found in muscle tissue; stores oxygen for rapid use during activity; high O2 affinity

Myoglobin structure

8 alpha-helices forming a compact globular structure; contains heme prosthetic group; iron binds oxygen reversibly (takes up oxygen at surplus and releases when needed at a site)

What is heme?

Protoporphyrin IX + Fe2+ (iron atom)

In the heme pocket, what does proximal histidine do?

His F8 directly coordinates Fe2+

In the heme pocket, what does distal histidine do?

His E7 stabilizes bound O2 and reduces CO binding

What does the heme pocket do?

Its hydrophobic pocket protects Fe2+ from oxidation

How does oxygen binding in myoglobin work?

There is a hyperbolic O2-binding curve that has a very high affinity for oxygen, making it function as an oxygen storage protein. There is no cooperativity (single binding site)

What is the physiological role of myoglobin?

Buffers oxygen supply in muscle, provides O2 during intense muscular activity, and is important in diving mammals

How does myoglobinuria work?

The muscle injury/rhabdomyolysis releases myoglobin into the blood and gets filtered into the urine, making it dark brown, and can cause acute kidney injury (diagnostic clue in crash injuries)

How does a myocardial infarction work?

Myoglobin is an early biomarker of a heart attack and rises within 2-3 hrs post-MI; not specific (also elevated in skeletal muscle injury) and now largely replaced by troponins in clinical practice

Basics of hemoglobin

Tetramer, variable O2 affinity, transport role

Hemoglobin overview

Major oxygen transport protein in red blood cells; 7 alpha-helices; tetramer (2 alpha + 2 beta chains -> adult HbA); each subunit has a heme group; 2,3-bisphosphoglycerate (2,3-BPG) -> holds subunits together; carries oxygen from lungs to tissues

What is the structure of hemoglobin?

Quaternary structure (heterotetramer); subunits interact via noncovalent forces; two conformations (T, R)

What is the T conformation?

Tense, low affinity for O2; because of its low O2 affinity, it doesn't really bind to oxygen and unloads it; ex: vein bc it is deoxygenated

What is the R conformation?

Relaxed, high affinity for O2; binds more to oxygen so it would load up more oxygen; ex: aorta bc it is oxygenated

What do both conformations have in common?

Has strong hydrophobic bonds and weak ionic & H-bonds

What is the oxygen binding curve?

Hemoglobin has a sigmoidal curve (S-shape) -> hemoglobin and there is a positive cooperativity; binding of first O2 increases affinity for others

Hill coefficient (nH)

Reflects the degree of cooperativity (binding of a molecule to one site influences the binding of subsequent molecules to other sites)

What is the nH for hemoglobin?

2.8

What is the nH for myoglobin?

1

Bohr effect

As pH decreases, O2 affinity also decreases because it unloads O2 from hemoglobin

What does CO2 do?

Forms carbaminohemoglobin -> stabilizes T state

What does 2,3-BPG do?

Binds beta-chains and lowers O2 affinity

Allosteric regulation of hemoglobin

Allosteric effectors fine-tune O2 delivery

What does exercise do?

More CO2 + H+ -> causes right shift -> enhanced O2 release

What does high altitude do?

As 2,3-BPG increases -> causes a right shift -> improves unloading

What does pregnancy do?

HbF (alpha 2 gamma 2) has higher O2 affinity -> facilitates maternal-fetal O2 transfer

Types of hemoglobin variants

HbA, HbA2, HbF

HbA

Alpha 2 beta 2; normal adult form

HbA2

Alpha 2 delta 2; minor adult form (2%)

HbF

Alpha 2 gamma 2; fetal hemoglobin, higher O2 affinity

Clinical use for hemoglobin variants

Persistence of HbF can heal sickle cell disease

HbS

Glu6Val mutation in beta-chain

Sickle cell disease

Deoxy-HbS polymerizes -> sickle shape; there's hemolytic anemia, vaso-occlusion, pain crises

Treatment for sickle cell disease

Hydroxyurea (increase of HbF), transfusions, gene therapy (cut out mutated parts)

Thalassemias

Caused by alpha- or beta-globin chain synthesis defects; imbalance causes ineffective erythropoiesis; microcytic anemia, skeletal deformities, splenomegaly

Treatment for thalassemias

Transfusions, iron chelation (remove excess iron), bone marrow transplant

CO poisoning

CO binds heme with 200x higher affinity than O2; stabilizes R state, prevents O2 release; symptoms: headache, dizziness, cherry-red skin

Treatment for CO poisoning

100% O2, hyperbaric (pressurized) O2

Methemoglobinemia

Fe2+ oxidized to Fe3+ (cannot bind O2); caused by drugs (benzocaine, dapsone), nitrates; cyanosis, chocolate-brown blood

Treatment for methemoglobinemia

Methylene blue

What is Hb regulated by that Mb is not?

pH, CO2, 2,3-BPG

What is the pharmacology link? (O2)

O2 delivery affects drug metabolism in the liver (hypoxia alters clearance); anesthetics can impact oxygen delivery; nitrites: induce methemoglobinemia -> therapeutic in cyanide poisoning

What are the key regulators of hemoglobin?

Bohr effect (H+), CO2 binding, 2,3-BPG levels; clinical: high altitude adaptation and pregnancy

Why is myoglobin not suitable for oxygen transport in blood?

Myoglobin is only used for storage purposes, releases oxygen when needed to, not in circulation, and is oxygen concentration dependent

How does 2,3-BPG shift the hemoglobin O2 curve?

Lowers O2 affinity

Why is HbF important in pregnancy?

Has higher affinity for O2 and can steal O2 from HbA

Which mutation causes sickle cell disease?

Glu -> Val