Acid-Base Chemistry of Peptides

Understanding pH-dependent protonation states and ionizable groups in amino acids

Contents

Why pH Matters for Peptides

The biological activity, solubility, stability, and even the three-dimensional structure of peptides are profoundly influenced by pH. Unlike simple small molecules, peptides contain multiple ionizable groups that can gain or lose protons (H⁺) depending on the pH of their environment.

Understanding acid-base chemistry is essential for:

🧬 Protein Function
Active sites depend on specific protonation states. Changing pH by just 0.5 units can completely abolish enzyme activity.
💊 Drug Design
Membrane permeability and receptor binding are pH-dependent. Charged peptides cannot cross lipid bilayers.
🔬 Experimental Design
Buffer selection, purification strategies, and storage conditions all require understanding pH effects.
⚗️ Chemical Synthesis
Protecting group strategies and coupling reactions depend on controlling protonation states during synthesis.
🎯 Learning Objectives

By the end of this guide, you will be able to:

  • Identify all ionizable groups in a peptide sequence
  • Predict protonation states at different pH values using pKa values
  • Apply the Henderson-Hasselbalch equation to calculate protonation ratios
  • Describe effects of pH on peptide properties and biological function
  • Use PepDraw's interactive pH slider to visualize protonation changes

Ionizable Groups in Amino Acids

In every peptide, there are three types of ionizable groups:

1. N-Terminus (α-amino group)

Every peptide has an amino group (–NH₃⁺ when protonated) at its N-terminus. This group acts as a base and can accept a proton.

N-terminus Equilibrium
–NH₃⁺ ⇌ –NH₂ + H⁺
Typical pKa ≈ 9.0
At pH < 9: Mostly protonated (–NH₃⁺, positive charge)
At pH > 9: Mostly deprotonated (–NH₂, neutral)

2. C-Terminus (α-carboxyl group)

Every peptide has a carboxyl group (–COOH when protonated) at its C-terminus. This group acts as an acid and can donate a proton.

C-terminus Equilibrium
–COOH ⇌ –COO⁻ + H⁺
Typical pKa ≈ 3.1
At pH < 3: Mostly protonated (–COOH, neutral)
At pH > 3: Mostly deprotonated (–COO⁻, negative charge)

3. Ionizable Side Chains

Seven amino acids have ionizable side chains. These residues dramatically affect peptide behavior:

Aspartic Acid (D)
–COOH ⇌ –COO⁻ + H⁺
pKa = 3.9
Negative charge at physiological pH (7.4)
Found in active sites, metal binding
Glutamic Acid (E)
–COOH ⇌ –COO⁻ + H⁺
pKa = 4.07
Negative charge at physiological pH
Longer side chain than Asp
Lysine (K)
–NH₃⁺ ⇌ –NH₂ + H⁺
pKa = 10.54
Positive charge at physiological pH
Common in DNA/membrane binding
Arginine (R)
Guanidinium: =NH₂⁺ ⇌ =NH + H⁺
pKa = 12.48
Always positive at physiological pH
Strongest base in proteins
Histidine (H)
Imidazole: =NH⁺ ⇌ =N + H⁺
pKa = 6.04
Can switch charge near physiological pH
Critical in catalytic mechanisms
Cysteine (C)
–SH ⇌ –S⁻ + H⁺
pKa = 8.3
Neutral at physiological pH
Forms disulfide bonds
Tyrosine (Y)
Phenol: –OH ⇌ –O⁻ + H⁺
pKa = 10.07
Neutral at physiological pH
Rarely ionized in proteins
⚠️ Important Note

Histidine is special! With a pKa around 6.0, histidine is the only amino acid that can switch between protonated and deprotonated states near physiological pH (7.4). This makes it incredibly important in enzyme catalysis, where it can act as both an acid and a base.

Understanding pKa Values

The pKa is the pH at which exactly 50% of molecules are protonated and 50% are deprotonated. It's the equilibrium point for the ionization reaction.

Key Concepts

Lower pKa = Stronger Acid
Aspartic acid (pKa = 3.9) readily donates a proton. It's deprotonated (negatively charged) at physiological pH.
Higher pKa = Stronger Base
Arginine (pKa = 12.48) readily accepts a proton. It's protonated (positively charged) at physiological pH.

The "2 pH Unit Rule"

A practical rule of thumb for predicting protonation states:

Quick Prediction Rules
  • pH < pKa - 2: Group is >99% protonated
  • pH = pKa: Group is exactly 50% protonated
  • pH > pKa + 2: Group is >99% deprotonated

Example: Lysine Side Chain (pKa = 10.54)

pH 7.0 (Cytoplasm)
pH < pKa - 2
>99% protonated: –NH₃⁺
Carries positive charge
pH 10.54 (Extreme)
pH = pKa
50% protonated, 50% deprotonated
Equal mixture
pH 13.0 (Strong Base)
pH > pKa + 2
>99% deprotonated: –NH₂
Neutral, no charge

The Henderson-Hasselbalch Equation

For precise calculations of protonation state, we use the Henderson-Hasselbalch equation. This fundamental relationship connects pH, pKa, and the ratio of protonated to deprotonated forms.

Henderson-Hasselbalch Equation
pH = pKa + log₁₀([A⁻]/[HA])
For bases (like amino groups):
pH = pKa + log₁₀([NH₂]/[NH₃⁺])

For acids (like carboxyl groups):
pH = pKa + log₁₀([COO⁻]/[COOH])

Using the Equation

Rearranging to find the fraction of deprotonated form:

Fraction deprotonated = 1 / (1 + 10^(pKa - pH))

Interactive Calculator

🧮 Henderson-Hasselbalch Calculator

Calculate the fraction of molecules in each protonation state:

Worked Example

📝 Example: Histidine at pH 7.4

What fraction of histidine side chains are protonated at physiological pH?

Given: Histidine pKa = 6.04, pH = 7.4

Solution:
Fraction deprotonated = 1 / (1 + 10^(6.04 - 7.4))
= 1 / (1 + 10^(-1.36))
= 1 / (1 + 0.044)
= 1 / 1.044
= 0.958 or 95.8%

Answer: At pH 7.4, about 96% of histidine side chains are deprotonated (neutral) and only 4% are protonated (positively charged). This is why histidine is often neutral at physiological pH.

Interactive pH Demonstration

See how different amino acids respond to pH changes in real-time. This demonstration shows the protonation state of each ionizable group across the pH range.

🎚️ Interactive pH Slider

Adjust the pH slider to see how protonation states change:

Acidic (0) Neutral (7) Basic (14)
🎯 Try This in PepDraw!

The pH slider in PepDraw's main app uses this exact chemistry to visualize protonation states in your peptide structures. As you adjust pH, you'll see:

  • Heteroatom labels change (NH₃⁺ → NH₂, COOH → COO⁻)
  • Net charge updates in real-time
  • Visual representation of ionization states
Try PepDraw's pH Visualization →

Visualize pH Effects on Your Peptides

Use PepDraw's interactive pH slider to see protonation states change in real-time. Watch as amino acids gain and lose protons with publication-quality visualization.

Launch PepDraw