Amino Acids as Acids and Bases: Zwitterions and pI

Every protein in your body is built from amino acids, and every amino acid is a small acid–base system in its own right. Each one carries an acidic group and a basic group on the same molecule, so it can gain or lose protons depending on the pH around it. That simple fact explains why amino acids have unusually high melting points, why proteins buffer your blood, and how scientists separate proteins in the laboratory.

The structure

A standard amino acid has a central carbon (the α-carbon) attached to four groups:

• an amino group, –NH₂ (basic)
• a carboxylic acid group, –COOH (acidic)
• a hydrogen atom
• a side chain, R, which differs from one amino acid to another

The simplest amino acid, glycine, has R = H: H₂N–CH₂–COOH.

Zwitterions

Here’s the twist. Because an amino acid has an acidic group and a basic group in the same molecule, a proton can move from one to the other. In the solid state and in water near neutral pH, the carboxylic acid gives its proton to the amino group:

H₂N–CH₂–COOH → H₃N⁺–CH₂–COO⁻

The result is a zwitterion (from the German Zwitter, meaning “hybrid”): a molecule with both a positive charge and a negative charge, but no overall charge.

This explains several properties that would be surprising for a small organic molecule:

• High melting points. Glycine decomposes above about 230 °C instead of melting at a low temperature, because solid amino acids are held together by strong ionic attractions between zwitterions, like a salt.
• Solubility in water and poor solubility in non-polar solvents.
• Large dipole moments.

How the charge changes with pH

An amino acid can exist in three main forms, depending on pH:

Condition	Form	Overall charge
Strongly acidic (low pH)	H₃N⁺–CHR–COOH	+1 (cation)
Near the isoelectric point	H₃N⁺–CHR–COO⁻	0 (zwitterion)
Strongly alkaline (high pH)	H₂N–CHR–COO⁻	−1 (anion)

• In acid, the –COO⁻ group picks up a proton to become –COOH. The molecule has a net positive charge.
• In alkali, the –NH₃⁺ group loses a proton to become –NH₂. The molecule has a net negative charge.

So amino acids are amphoteric: they react with both acids and bases. See amphoteric substances.

Two pKa values

Treat the fully protonated form of glycine as a diprotic acid:

H₃N⁺–CH₂–COOH ⇌ H₃N⁺–CH₂–COO⁻ + H⁺ pKa₁ ≈ 2.34 H₃N⁺–CH₂–COO⁻ ⇌ H₂N–CH₂–COO⁻ + H⁺ pKa₂ ≈ 9.60

• pKa₁ belongs to the carboxylic acid group. It’s much lower than for ethanoic acid (4.76), because the nearby –NH₃⁺ group pulls electron density away and stabilises the –COO⁻.
• pKa₂ belongs to the –NH₃⁺ group.

Each pKa marks the pH where two forms are present in equal amounts, as in any polyprotic acid.

The isoelectric point (pI)

The isoelectric point, pI, is the pH at which the amino acid has no net charge: the zwitterion dominates, and any small amounts of cation and anion balance each other.

For an amino acid with a non-ionisable side chain, the pI is the average of the two pKa values:

pI = (pKa₁ + pKa₂) ÷ 2

For glycine: pI = (2.34 + 9.60) ÷ 2 = 5.97

For alanine (pKa₁ 2.34, pKa₂ 9.69): pI = 6.02.

Amino acids with ionisable side chains

Some amino acids have an extra acidic or basic group in their side chain, which changes the pI.

Acidic side chains: aspartic acid and glutamic acid have an extra –COOH. Their pI values are low (about 2.8 and 3.2), because they need a lot of acid to cancel their extra negative charge. For these, the pI is the average of the two lowest pKa values.

Example: aspartic acid, pKa values 1.88 (α-COOH), 3.65 (side-chain COOH), 9.60 (α-NH₃⁺). pI = (1.88 + 3.65) ÷ 2 = 2.77.

Basic side chains: lysine, arginine and histidine have extra nitrogen groups. Their pI values are high (lysine about 9.7, arginine about 10.8). For these, the pI is the average of the two highest pKa values.

Example: lysine, pKa values 2.18, 8.95 (α-NH₃⁺), 10.53 (side-chain NH₃⁺). pI = (8.95 + 10.53) ÷ 2 = 9.74.

Histidine is special. Its side chain (an imidazole ring) has a pKa of about 6.0, close to physiological pH. That lets it gain or lose a proton easily in the body, making it an excellent proton shuttle in enzyme active sites and an important buffer in proteins like haemoglobin.

Amino acid	Side chain type	pI (approx.)
Aspartic acid	acidic	2.8
Glutamic acid	acidic	3.2
Glycine	neutral	6.0
Alanine	neutral	6.0
Histidine	basic (weak)	7.6
Lysine	basic	9.7
Arginine	basic	10.8

Why the pI matters

Electrophoresis

In electrophoresis, molecules move through a gel or paper under an electric field. An amino acid or protein at a pH below its pI is positively charged and moves towards the negative electrode. Above its pI, it’s negatively charged and moves towards the positive electrode. At its pI, it doesn’t move.

So at pH 6, glycine (pI 6.0) stays put, lysine (pI 9.7) moves towards the negative electrode, and glutamic acid (pI 3.2) moves towards the positive electrode. This is how mixtures of amino acids can be separated and identified.

Isoelectric focusing takes this further: proteins migrate through a pH gradient until each stops at its own pI, separating proteins that differ by as little as 0.01 pH units.

Protein solubility

Proteins are least soluble at their pI, because with no net charge, the molecules don’t repel each other and tend to clump together. This is used to precipitate proteins, and it’s part of why milk curdles when it becomes acidic: casein, the main milk protein, has a pI of about 4.6. As bacteria produce lactic acid and the pH falls towards 4.6, casein loses its charge and clumps, which is how yoghurt and cheese form.

Buffering in the body

The ionisable groups of amino acids in proteins, especially histidine, help buffer blood and cells against pH changes. See the blood buffer system.

Protein shape

The charges on amino acid side chains form ionic attractions (salt bridges) that help hold a protein in its folded shape. Change the pH far enough and those charges change, which can unfold the protein (denaturation). That’s one reason enzymes have an optimum pH.

Worked example: what charge at pH 7.4?

Predict the main form and overall charge of glycine, lysine and aspartic acid at blood pH (7.4).

• Glycine (pKa 2.34, 9.60): pH 7.4 is between the two, so the zwitterion dominates. Charge ≈ 0.
• Lysine (pKa 2.18, 8.95, 10.53): both amino groups are still protonated (pH below 8.95 and 10.53), the carboxylate is deprotonated. Charge ≈ +1.
• Aspartic acid (pKa 1.88, 3.65, 9.60): both carboxyl groups are deprotonated, the amino group is protonated. Charge ≈ −1.

Key takeaways

• Amino acids contain an acidic –COOH and a basic –NH₂, so they exist as zwitterions near neutral pH.
• They’re cations at low pH, zwitterions near the isoelectric point, and anions at high pH.
• For simple amino acids, pI = (pKa₁ + pKa₂) ÷ 2; for acidic or basic side chains, average the two relevant pKa values.
• The pI controls movement in electrophoresis and protein solubility, and explains milk curdling at pH 4.6.
• For the equilibrium ideas behind pKa, see Ka and pKa.