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Hemoglobin &
Oxygen Binding

Explore how hemoglobin's cooperative binding delivers oxygen to tissues and how pH, temperature, and allosteric effectors shift the oxygen-dissociation curve.

Biochemistry

What Is Hemoglobin?

Hemoglobin (Hb) is a tetrameric protein found in red blood cells. It consists of four polypeptide subunits: two \(\alpha\) chains and two \(\beta\) chains, each containing a heme group with an iron (\(\text{Fe}^{2+}\)) center that reversibly binds one molecule of \(\text{O}_2\).

4 Heme Groups
Each subunit holds one heme with an \(\text{Fe}^{2+}\) ion capable of binding \(\text{O}_2\).
Quaternary Structure
The \(\alpha_2\beta_2\) tetramer enables cooperative interactions between subunits.
Reversible Binding
\(\text{Hb} + 4\,\text{O}_2 \rightleftharpoons \text{Hb(O}_2)_4\): bind in lungs, release in tissues.

T-State vs R-State

Hemoglobin switches between two conformations. The T (tense) state has low oxygen affinity and predominates in deoxygenated blood. The R (relaxed) state has high oxygen affinity and is stabilized when \(\text{O}_2\) is bound.

T-State (Tense)
Low \(\text{O}_2\) affinity
 Constrained subunits Deoxy-hemoglobin
\(+\text{O}_2\)
 R-State (Relaxed)
High \(\text{O}_2\) affinity
 Open conformation Oxy-hemoglobin

Why Cooperative Binding Matters

Without cooperativity, hemoglobin would behave like myoglobin: a simple hyperbolic curve. Instead, \(\text{O}_2\) binding follows a sigmoidal (S-shaped) curve, meaning hemoglobin loads efficiently at high \(pO_2\) (lungs) and unloads efficiently at low \(pO_2\) (tissues).

1
First \(\text{O}_2\) binds slowly: T-state has low affinity, so binding requires high \(pO_2\).
2
Conformational change: \(T \rightarrow R\) transition loosens neighboring subunits.
3
Remaining \(\text{O}_2\) molecules bind rapidly: affinity increases with each binding event.
4
Sigmoidal saturation curve: steep rise in the physiological range maximizes \(\text{O}_2\) delivery.

The Oxygen-Dissociation Curve

The Hill Equation

The Hill equation quantifies the degree of cooperativity in ligand binding:

\(\theta\)
Fractional saturation (0 to 1)
\(P_{50}\)
\(pO_2\) at 50% saturation (~26 mmHg for Hb)
\(n\)
Hill coefficient: \(n \approx 2.8\) for Hb (positive cooperativity)

The Bohr Effect: pH Shifts the Curve

Active tissues produce \(\text{CO}_2\), which lowers pH. Protons (\(\text{H}^+\)) bind to specific residues on hemoglobin (notably His-146 on the \(\beta\) chain), stabilizing the T-state and decreasing \(\text{O}_2\) affinity. This is the Bohr effect.

Hemoglobin vs Myoglobin

Hemoglobin Myoglobin
Structure Tetramer (\(\alpha_2\beta_2\)) Monomer
Binding sites 4 heme groups 1 heme group
Curve shape Sigmoidal Hyperbolic
Cooperativity Yes (\(n \approx 2.8\)) No (\(n = 1\))
\(P_{50}\) ~26 mmHg ~2.8 mmHg
Function \(\text{O}_2\) transport in blood \(\text{O}_2\) storage in muscle
Bohr effect Yes No

Allosteric Effectors of Hemoglobin

Several molecules shift the oxygen-dissociation curve by stabilizing the T or R state:

\(\text{H}^+\) (pH)
Low pH stabilizes T-state: curve shifts right (Bohr effect).
\(\text{CO}_2\)
Binds N-termini as carbamate, stabilizes T-state: curve shifts right.
2,3-BPG
Binds central cavity of deoxy-Hb, lowers affinity: curve shifts right.
Temperature
Higher temperature weakens \(\text{Hb-O}_2\) bonds: curve shifts right.

Right Shift vs Left Shift

Right Shift (decreased affinity)
Increased: \(\text{H}^+\), \(\text{CO}_2\), 2,3-BPG, temperature.
Effect: \(P_{50}\) increases, \(\text{O}_2\) is released more readily to tissues.
Left Shift (increased affinity)
Decreased: \(\text{H}^+\), \(\text{CO}_2\), 2,3-BPG, temperature.
Effect: \(P_{50}\) decreases, Hb holds \(\text{O}_2\) more tightly (e.g., fetal Hb).

How to Analyze Curve Shifts

1
Identify the variable: Is pH, \(\text{CO}_2\), temperature, or 2,3-BPG changing?
2
Determine the direction: Is the variable increasing or decreasing?
3
Apply the rule: Increased \(\text{H}^+\), \(\text{CO}_2\), temperature, or 2,3-BPG shifts the curve right (lower affinity). Decreases shift it left.
4
Interpret \(P_{50}\): Right shift = higher \(P_{50}\). Left shift = lower \(P_{50}\).
5
Predict the effect: Right shift means more \(\text{O}_2\) unloaded at tissues. Left shift means tighter \(\text{O}_2\) retention.

Example 1: Exercising Muscle

Scenario: During intense exercise, muscle tissue \(pO_2\) drops to ~20 mmHg, pH falls to ~7.2, and local \(\text{CO}_2\) and temperature rise. What happens to \(\text{O}_2\) delivery?

1
Variables changing: pH down, \(\text{CO}_2\) up, temperature up, 2,3-BPG up.
2
All four shift the curve right: \(P_{50}\) increases from ~26 to ~35 mmHg.
3
At \(pO_2 = 20\) mmHg: Normal curve gives ~30% saturation; right-shifted gives ~20%.
4
Result: More \(\text{O}_2\) is released per hemoglobin molecule, increasing delivery to active muscle.

Example 2: Carbon Monoxide Poisoning

Scenario: CO binds hemoglobin with ~240 times higher affinity than \(\text{O}_2\). How does this affect the remaining subunits and the dissociation curve?

Example 3: Fetal Hemoglobin (HbF)

Question: HbF has \(\alpha_2\gamma_2\) subunits. The \(\gamma\) chain binds 2,3-BPG more weakly than the adult \(\beta\) chain. How does this affect fetal \(\text{O}_2\) uptake?

1
Less 2,3-BPG binding: the T-state is less stabilized in HbF.
2
Curve shifts left: HbF has a lower \(P_{50}\) (~19 mmHg) and higher \(\text{O}_2\) affinity.
3
At placental \(pO_2\) (~40 mmHg): maternal HbA is ~75% saturated, while fetal HbF reaches ~85%.
4
Net effect: \(\text{O}_2\) flows from maternal Hb (lower affinity) to fetal HbF (higher affinity) down the affinity gradient.

Common Mistakes

✕ Confusing pH Direction
Lower pH (more acidic) shifts the curve right, not left. Remember: acids are produced in active tissue where \(\text{O}_2\) needs to be released.
✕ Mixing Up Hb and Mb Curves
Myoglobin is hyperbolic (\(n=1\)), hemoglobin is sigmoidal (\(n \approx 2.8\)). Myoglobin has higher affinity but no cooperativity.
✕ Forgetting CO's Dual Effect
CO doesn't just block sites: it also shifts the curve left for remaining subunits, making \(\text{O}_2\) release even harder.

Summary: Factors Affecting \(\text{O}_2\) Binding

Factor Right Shift (Release) Left Shift (Retain)
pH Decrease (acidosis) Increase (alkalosis)
\(\text{CO}_2\) Increase Decrease
Temperature Increase Decrease
2,3-BPG Increase Decrease
CO N/A Left shift + blocks sites
Fetal Hb N/A Left shift (low BPG binding)

Question 1 of 5

The oxygen-dissociation curve for hemoglobin is sigmoidal rather than hyperbolic because of:

Question 2 of 5

A patient has a blood pH of 7.2 (normal: 7.4). What happens to hemoglobin's \(P_{50}\)?

Question 3 of 5

Which statement best explains why fetal hemoglobin (HbF) has a higher oxygen affinity than adult hemoglobin (HbA)?

Question 4 of 5

In the Hill equation \(\theta = \frac{(pO_2)^n}{(P_{50})^n + (pO_2)^n}\), a Hill coefficient \(n = 1\) indicates:

Question 5 of 5

Carbon monoxide (CO) poisoning is especially dangerous because CO: