Allosteric Effects > > Proteins

HEMOGLOBIN ALLOSTERIC EFFECTS

OVERVIEW

DEFINITIONS

Bohr effect

• Low pH enhances hemoglobin dissociation

2, 3 Bisphosphoglycerate (BPG)

• localized in cells

• Decreases hemoglobin's oxygen affinity

DISSOCIATION CURVE

• % vs. oxygen (torr)

• produces sigmoidal binding curve

Bohr Effect

• Decrease in blood pH shifts curve to the right

• Hemoglobin requires greater pO2 in peripheral tissues to reach 50% saturation

• Lowering pH decreases hemoglobin's oxygen affinity

2,3 BPG

• Hemoglobin without 2,3 BPG: shifts curve to the left (hyperbolic like )

• Adding 2,3 BPG: shifts curve back to the right.

1 / 7 BOHR EFFECT

Beta subunit

• Aspartate (-) and (+)

T-form: histidine pKa = 8.0 (side chains close to each other)

• T-form favored when blood pH --> has a high affinity for H+

R-form: histidine pKa drops to 7.1 (side chains move apart)

• Histidine loses H+

• R-form favored when blood pH is high and H+ is low

Carbon dioxide

• CO2 + H2O --> H2CO3 (carbonic ) --> HCO3- () + H+ (reversible)

• catalyzes formation

• Carbonic acid spontaneously loses to form bicarbonate

• Bicarbonate = blood buffer

• Increase in CO2 --> lowers blood pH --> favors T-form hemoglobin

Carbaminohemoglobin: CO2 binds N-terminal amino in hemoglobin

2,3 BPG

• Has strong negative charge: binds central cavity in hemoglobin

• Stabilizes the T-form (only binds T-form)

CARBON MONOXIDE

• Binds center with 220 times the affinity of O2 (irreversible)

• Permanently increases oxygen affinity of remaining groups for oxygen

• Decreases oxygen release in peripheral tissues

CLINICAL CORRELATION

Acetazolamide

2 / 7 • Carbonic anhydrase inhibitor used to treat altitude sickness

• Increases bicarbonate excretion by kidneys

• Makes blood more acidic, promotes oxygen release in peripheral tissues

High altitude conditions

• Individuals adapted to high altitude produce more 2,3 BPG

• Favors T-form hemoglobin and O2 release: more efficient O2 delivery

Tobacco smoke

• Smokers have elevated blood CO: hinders O2 delivery

• Can produce tissue

FULL-LENGTH TEXT

• Here we will learn about the Bohr effect, and discuss the effects of 2,3 bisphosphoglycerate and on hemoglobin.

• To begin, start a table to list out each of the key topics we'll learn.

- Bohr effect, which describes the effects of pH and on hemoglobin binding.

- 2,3 Bisphosphoglycerate (BPG), a molecule localized in red blood cells that decreases hemoglobin's affinity for oxygen.

- Carbon monoxide, which increases hemoglobin's oxygen affinity and can produce toxic effects in the body.

• As a review, draw hemoglobin's sigmoidal dissociation curve.

• Label the x-axis partial pressure and the y-axis percent saturation.

Now, let's illustrate the first allosteric effect: the Bohr Effect.

• Draw another sigmoidal curve to the right of the first one.

3 / 7 • Show that a decrease in blood pH, shifts hemoglobin's dissociation curve to the right.

• What does this mean?

- Extend the horizontal line that demarcates 50% saturation.

- Show that it intersects the second curve at a greater partial pressure of oxygen: lowering pH decreases hemoglobin's affinity for oxygen.

• To better understand this, draw the three-dimensional structure of hemoglobin: with two alpha subunits and two beta subunits.

Let's take a closer look at a beta-subunit.

• Within it draw two functional groups: that of aspartate (negative) and histidine (positive).

• Indicate that in T-form hemoglobin, these side chains are close to each other, which raises the pKa of histidine to 8.0.

- Thus, hemoglobin has a high affinity for in the deoxygenated state (high pKa means a high proton affinity).

Now, let's draw these groups in R-form hemoglobin.

• Show that they are farther apart, and that the pKa of histidine drops to 7.1.

- As we have seen, the transition between T-form and R-form hemoglobin causes shifts in conformations throughout the entire .

• Show that because its pKa decreases, histidine loses a proton.

• Thus, write that R-form is favored when blood pH is high, and the proton concentration is low.

• Now, write that T-form is favored when the blood pH is low, and the proton concentration is high.

- Thus, a low pH enhances oxygen dissociation and shifts hemoglobin's dissociation curve to the right.

Now, we know that a low pH enhances oxygen dissociation. But what produces low blood pH in the first place?

- Carbon dioxide!

• Write out the following equation:

4 / 7 - Carbon dioxide plus water reversibly converts to carbonic acid.

• Show that the enzyme carbonic anhydrase catalyzes this reaction.

• Next, indicate that carbonic acid spontaneously loses its proton to form bicarbonate.

- Thus bicarbonate releases protons into the blood stream.

• Indicate that bicarbonate functions as a buffer in the blood.

• As a clinical correlation, write that acetazolamide is a carbonic anhydrase inhibitor that is often used to treat altitude sickness.

- How does it work? It produces an increase in bicarbonate excretion by the kidneys, via a mechanism we will not cover here.

- As a result, it makes the blood more acidic, and promotes the release of oxygen in the peripheral tissues.

• Finally, write that an increase in carbon dioxide in the body lowers blood pH and favors T-form hemoglobin.

- This facilitates hemoglobin's physiologic function in the body.

• Note that carbon dioxide can also form by binding to N-terminal amino acids in hemoglobin. We will not discuss this, here.

Now that we have learned the Bohr effect, let's move on to 2,3-bisphosphoglycerate (BPG), a molecule synthesized by red blood cells.

Our current sigmoidal dissociation curve already accounts for the allosteric effects of normal 2,3-BPG levels in red blood cells.

• To visualize 2,3-BPG's effects, draw a hyperbolic curve to the left of the hemoglobin curve.

- It should resemble myoglobin's dissociation curve.

• Indicate that this curve represents hemoglobin without 2,3-BPG.

- Thus, without 2,3-BPG, hemoglobin's oxygen affinity increases dramatically.

• Show that adding 2,3-BPG shifts the curve back to the right.

5 / 7 How does 2,3-BPG lower hemoglobin's oxygen affinity? Let's illustrate this, now.

• To do this, take a closer look at the center of our hemoglobin molecule: the cavity created at the intersection of all four subunits.

• Draw histidine residues at the periphery of each of the beta subunits.

• Show that these are positively charged; they repel each other.

• Now, importantly, label this diagram the T-form (deoxygenated hemolgobin).

Now, let's add 2,3-BPG.

• Draw a 2,3-BPG molecule within this central cavity.

• Indicate that it has a strong negative charge, which stabilizes the T-form.

- Other positively charged amino acid side chains also bind 2,3 BPG here, but we won't draw all of them.

• Now, write that 2,3-BPG only binds the T-form.

- It encourages oxygen dissociation, and facilitates oxygen delivery!

• As a clinical correlation, indicate that individuals that have adapted to high altitude conditions produce more BPG.

- Why? More BPG favors T-form hemoglobin and oxygen release; it allows hemoglobin to deliver more oxygen to the peripheral tissues.

Finally, let's illustrate carbon monoxide.

• Draw a simplified hemoglobin iron center.

• Show that carbon monoxide irreversibly binds it.

- We draw it at an angle because a distal histidine causes it to bend.

• Write that carbon monoxide binds iron with an affinity 220 times greater than oxygen!

6 / 7 • Indicate that by irreversibly binding iron, it permanently increases the oxygen affinity of the remaining heme groups for oxygen.

- Thus, it pushes the hemoglobin dissociation curve to the left.

What are the physiological consequences?

• Indicate that carbon monoxide leads to decreased oxygen release in the peripheral tissues.

• As a clinical correlation, write that people who regularly smoke tobacco have elevated levels of carbon monoxide in their blood, which hinders hemoglobin's ability to deliver oxygen.

- Thus, a consequence of elevated carbon monoxide is tissue hypoxia.

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