Lecture 2.2: Soluble Transport Proteins

What is the primary function of hemoglobin?

Hemoglobin transports O₂ from the lungs to tissues through the bloodstream.

What is the primary function of myoglobin?

Myoglobin stores O₂ in muscle and releases it when muscle pO₂ becomes very low.

What is the subunit composition of adult hemoglobin?

Adult hemoglobin is an α₂β₂ tetramer containing two alpha subunits and two beta subunits.

How many O₂ molecules can one hemoglobin molecule bind?

Four O₂ molecules because each of its four subunits contains one heme group.

How many O₂ molecules can one myoglobin molecule bind?

One O₂ molecule because myoglobin has one subunit and one heme group.

What does pO₂ represent?

pO₂ is the partial pressure of oxygen, which represents the amount of available O₂ in an environment.

What is fractional saturation, θ?

Fractional saturation is the fraction of a protein’s O₂-binding sites that are occupied. A θ of 1 means all sites are occupied.

What shape is the myoglobin O₂-binding curve?

Hyperbolic because myoglobin has one binding site and does not show cooperativity.

What shape is the hemoglobin O₂-binding curve?

Sigmoidal, or S-shaped, because hemoglobin displays positive cooperativity.

Why is hemoglobin more effective than myoglobin at delivering O₂?

Hemoglobin’s cooperative binding lets it bind O₂ strongly in the lungs and release a large amount in tissues. Myoglobin holds O₂ too tightly at normal tissue pO₂.

Why does myoglobin release relatively little O₂ between the lungs and resting tissues?

Myoglobin has very high O₂ affinity and remains highly saturated at both lung and resting-tissue pO₂.

Why would lowering myoglobin’s O₂ affinity not make it an ideal transport protein?

Very low affinity could improve unloading but would also reduce O₂ loading in the lungs. Hemoglobin solves this by changing affinity cooperatively.

What is P₅₀?

P₅₀ is the pO₂ at which a protein is 50% saturated with O₂.

What does a low P₅₀ indicate?

A low P₅₀ indicates high O₂ affinity because little O₂ is needed for 50% saturation.

What does a high P₅₀ indicate?

A high P₅₀ indicates low O₂ affinity because more O₂ is needed for 50% saturation.

What does a right shift of the hemoglobin O₂-binding curve mean?

Hemoglobin has lower O₂ affinity, a higher P₅₀, and releases O₂ more easily.

What does a left shift of the hemoglobin O₂-binding curve mean?

Hemoglobin has higher O₂ affinity, a lower P₅₀, and holds O₂ more tightly.

Where does hemoglobin normally load O₂?

In the lungs, where pO₂ is high.

Where does hemoglobin normally unload O₂?

In tissues, where pO₂ is lower, especially in rapidly metabolizing tissues.

What is the T state of hemoglobin?

The tense, or T, state is the low-affinity and usually deoxygenated form of hemoglobin.

What is the R state of hemoglobin?

The relaxed, or R, state is the high-affinity and usually oxygenated form of hemoglobin.

What is cooperativity?

Cooperativity occurs when ligand binding at one subunit changes the affinity of other subunits for that ligand.

What is positive cooperativity?

Positive cooperativity means binding of one ligand increases the protein’s affinity for additional molecules of the same ligand.

How does hemoglobin demonstrate positive cooperativity?

Binding of one O₂ promotes the T-to-R transition, making the remaining subunits bind O₂ more easily.

How does the affinity of the last O₂-binding site compare with the first?

The last O₂ binds with approximately 100 times greater affinity than the first.

Why does cooperativity produce a sigmoidal curve?

Hemoglobin begins in a low-affinity state, but its affinity increases as additional O₂ molecules bind.

What happens to Fe²⁺ when O₂ binds to heme?

Fe²⁺ moves into the plane of the porphyrin ring.

Does Fe³⁺ or Fe²⁺ normally bind O₂ in functional hemoglobin?

Fe²⁺ normally binds O₂. Fe³⁺ forms methemoglobin and cannot bind O₂ normally.

What is the proximal histidine?

The proximal histidine, His F8, directly coordinates Fe²⁺ and connects the heme to the F helix.

What happens to the proximal histidine during O₂ binding?

It moves with Fe²⁺ toward the heme plane and pulls the F helix with it.

What happens to the F helix during the T-to-R transition?

The F helix shifts position and helps transmit the structural change throughout hemoglobin.

How is the movement of one heme communicated to other hemoglobin subunits?

F-helix movement rearranges noncovalent interactions at the subunit interfaces.

Which subunit interfaces are especially repositioned during the T-to-R transition?

The α₁-β₂ and α₂-β₁ interfaces.

Are new covalent bonds formed between hemoglobin subunits during the T-to-R transition?

No. Existing noncovalent interactions, such as hydrogen bonds and ionic interactions, are rearranged.

Does the T-to-R transition change hemoglobin’s primary structure?

No. The amino acid sequence remains unchanged.

Which structural levels change during the T-to-R transition?

Tertiary structure within the subunits and quaternary structure between the subunits change.

How does the central cavity differ between the T and R states?

The central cavity is larger in the T state and becomes smaller in the R state.

Why does increasing O₂ tension promote the R state?

More O₂ binds to heme groups, shifting hemoglobin toward the oxygenated, high-affinity R state.

Which statements are correct about the T-to-R transition?

Noncovalent interactions at the α₁-β₂ and α₂-β₁ interfaces are repositioned, and increased O₂ tension promotes the transition.

Why is the statement “Fe³⁺ moves into the heme plane” incorrect?

Functional hemoglobin uses Fe²⁺, not Fe³⁺, for reversible O₂ binding.

What is allostery?

Allostery occurs when binding at one site changes the structure, activity, or affinity of another site on the same protein.

What is an allosteric effector?

An allosteric effector is a molecule that binds to a protein and changes its structure, activity, or ligand affinity.

What is a positive allosteric effector?

A positive allosteric effector increases ligand affinity or protein activity.

What is a negative allosteric effector?

A negative allosteric effector decreases ligand affinity or protein activity.

What is a homotropic allosteric effector?

A homotropic effector is the same molecule as the primary ligand.

What is a heterotropic allosteric effector?

A heterotropic effector is different from the primary ligand.

How is O₂ classified as an effector of hemoglobin?

O₂ is a positive homotropic allosteric effector.

How are H⁺, CO₂, and 2,3-BPG classified as hemoglobin effectors?

They are negative heterotropic allosteric effectors because they are not O₂ and they decrease O₂ affinity.

What is the main purpose of a Hill plot?

A Hill plot detects and quantitatively assesses cooperative ligand binding.

What is plotted on the x-axis of a Hill plot?

The logarithm of ligand concentration, such as log[pO₂].

What is plotted on the y-axis of a Hill plot?

log[θ divided by 1 minus θ].

What does the slope of a Hill plot represent?

The slope represents the Hill coefficient, nH, which measures the degree of cooperativity.

What does nH = 1 mean?

There is no cooperativity, and the binding sites behave independently.

What does nH greater than 1 mean?

It indicates positive cooperativity, as seen in hemoglobin.

What does nH less than 1 mean?

It indicates negative cooperativity, meaning ligand binding makes additional binding less favorable.

Does nH = 1 necessarily mean that a protein has only one binding site?

No. A protein may have multiple independent binding sites and still have nH = 1.

Can the Hill coefficient exceed the total number of binding sites?

No. The number of interacting binding sites places an upper limit on the Hill coefficient.

What information does the Hill plot x-intercept provide?

At θ = 0.5, the x-intercept corresponds to log(P₅₀), or the apparent dissociation value.

How does higher temperature affect hemoglobin?

It favors the T state, lowers O₂ affinity, shifts the curve right, and promotes O₂ release.

How does lower temperature affect hemoglobin?

It increases O₂ affinity and shifts the curve to the left.

How does lower pH affect hemoglobin?

It favors the T state, lowers O₂ affinity, and shifts the curve to the right.

How does higher pH affect hemoglobin?

It favors the R state, increases O₂ affinity, and shifts the curve to the left.

What is the Bohr effect?

The Bohr effect is the decrease in hemoglobin’s O₂ affinity caused by increased H⁺ and CO₂.

Why does the Bohr effect help active tissues?

Active tissues produce acid and CO₂, causing hemoglobin to release more O₂ where it is needed.

What conditions are found in rapidly metabolizing tissues?

Higher temperature, higher CO₂, lower pH, lower pO₂, and often increased 2,3-BPG.

What conditions generally favor O₂ loading in the lungs?

High pO₂, lower CO₂, higher pH, and lower temperature compared with active tissues.

What are the three major ways CO₂ is transported in blood?

Dissolved CO₂, bicarbonate, and CO₂ attached directly to hemoglobin.

In what form is most CO₂ transported in blood?

Most CO₂ is transported as bicarbonate, HCO₃⁻.

What reaction does carbonic anhydrase accelerate?

CO₂ plus H₂O reversibly forms H₂CO₃, which reversibly forms H⁺ plus HCO₃⁻.

How does bicarbonate formation promote O₂ release?

It produces H⁺, lowering pH and contributing to the Bohr effect.

Where does CO₂ bind directly to hemoglobin?

CO₂ binds to the amino termini of hemoglobin subunits and forms carbamate groups.

How does direct CO₂ binding affect hemoglobin?

Carbamate formation creates additional ionic interactions that stabilize the T state.

Approximately how much CO₂ is transported directly by hemoglobin?

Approximately 15% of CO₂ is transported directly by hemoglobin.

What is 2,3-BPG?

2,3-BPG is a negatively charged byproduct of glycolysis that regulates hemoglobin’s O₂ affinity.

Where does 2,3-BPG bind hemoglobin?

It binds in the positively charged central cavity of deoxyhemoglobin.

Which hemoglobin state does 2,3-BPG stabilize?

The low-affinity T state.

How does 2,3-BPG affect the O₂-binding curve?

It lowers O₂ affinity, increases P₅₀, and shifts the curve to the right.

Why does 2,3-BPG bind less effectively to the R state?

The central cavity becomes smaller in the R state, leaving less space for 2,3-BPG.

Why is 2,3-BPG useful during exercise?

It promotes O₂ unloading to tissues that are rapidly using oxygen.

Which combination most strongly favors O₂ release?

Increased temperature, increased CO₂, decreased pH, and increased 2,3-BPG.

How does the body use 2,3-BPG to adapt to high altitude?

Red blood cells increase 2,3-BPG, lowering hemoglobin affinity and improving O₂ unloading to tissues.

At high altitude after 2,3-BPG adaptation, does hemoglobin have higher or lower O₂ affinity?

Hemoglobin has lower O₂ affinity.

In which direction does increased 2,3-BPG shift the curve at high altitude?

It shifts the curve to the right.

At high altitude, hemoglobin has what affinity for O₂ and which curve shift?

Hemoglobin has lower O₂ affinity, and the curve shifts to the right.

What is the trade-off of increasing 2,3-BPG at high altitude?

Hemoglobin loads slightly less O₂ in the lungs but releases a greater fraction of O₂ in tissues.

How does the His143-to-Ser change affect 2,3-BPG binding?

It removes a positively charged histidine and weakens attraction to negatively charged 2,3-BPG.

Why does fetal hemoglobin have higher O₂ affinity than adult hemoglobin?

Fetal hemoglobin binds 2,3-BPG less strongly, so its T state is less stabilized.

How is the fetal hemoglobin-binding curve positioned relative to adult hemoglobin?

The fetal hemoglobin curve is shifted to the left.

Why must fetal hemoglobin have higher O₂ affinity than maternal hemoglobin?

The higher affinity allows fetal blood to obtain O₂ from maternal blood across the placenta.

How do high-altitude adaptation and fetal hemoglobin affect O₂ affinity differently?

High-altitude adaptation increases BPG and shifts adult hemoglobin right, while fetal hemoglobin binds BPG weakly and is shifted left.

What determines whether an amino acid substitution changes a protein’s pI?

The substitution must add, remove, or change a charged side chain.

Why can the Leu-to-Pro substitution in Hb destabilize hemoglobin?

Proline restricts backbone movement and can break or bend an alpha helix.

Why are mutations near the heme pocket especially likely to be harmful?

They can directly change iron coordination, O₂ binding, oxidation state, or heme stability.

Why are mutations at hemoglobin subunit interfaces especially likely to be harmful?

They can disrupt tetramer stability, cooperativity, and communication between O₂-binding sites.

Why may a surface mutation be less harmful than a buried mutation?

Surface substitutions are less likely to disrupt the hydrophobic core, heme pocket, or subunit interfaces.

Can a mutation alter hemoglobin function without changing its pI?

Yes. An uncharged substitution can disrupt folding, hydrogen bonding, heme binding, or subunit interactions without changing net charge.

Can a mutation alter pI without causing severe disease?

Yes. A surface charge change may alter migration on an IEF gel without greatly disrupting hemoglobin structure or function.