Acid-Base Chemistry of Peptides

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)

⚠️ Terminal Modifications

Many peptides have modified termini that are NOT ionizable:

Acetylated N-terminus (Ac-): CH₃-CO-NH- (not ionizable, no charge contribution)
Amidated C-terminus (-NH₂): -CO-NH₂ (not ionizable, no charge contribution)

These modifications are extremely common in therapeutic peptides (e.g., GLP-1 analogs) and natural hormones (e.g., oxytocin). When calculating charge, exclude modified termini from your calculations. PepDraw's app includes options for these modifications and automatically adjusts charge calculations.

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

💡 Key Insight: Two pH Sensors Near Physiological pH

While histidine is famous for pH-sensing in enzyme active sites, it shares this role with cysteine. Both have pKa values close to physiological pH, making them exquisitely sensitive to pH changes in biological systems:

Histidine (pKa 6.04)

At pH 7.4: ~4% protonated (+)

ΔpH from pKa: +1.36 units

Mostly: Neutral with slight positive character

Cysteine (pKa 8.3)

At pH 7.4: ~11% deprotonated (−)

ΔpH from pKa: −0.9 units

Mostly: Neutral with slight negative character

Critical insight: Cysteine is actually closer to physiological pH than histidine (0.9 vs 1.36 pH units), making it highly responsive to microenvironmental pH changes. While cysteine is best known for disulfide cross-linking, its thiol group (–SH/–S⁻) plays crucial roles in:

Catalytic mechanisms: Thiolate anion (S⁻) is a powerful nucleophile in cysteine proteases
Redox sensing: pH affects thiol reactivity with peroxides and disulfides
Metal coordination: pH-dependent binding of Zn²⁺, Fe²⁺ in metalloproteins
Microenvironment sensing: Detects pH gradients near membranes and in organelles

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:

pKa of ionizable group:

pH of solution:

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:

pH: 7.0

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 →

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