Amino Acids and Peptides: Zwitterions, pI and the Peptide Bond
Almost every property of an amino acid follows from the fact that it carries an acid and a base in the same molecule.
The zwitterion
An amino acid has a carboxylic acid and an amine on the same molecule. The acid protonates the amine internally, so at intermediate pH the molecule carries both a positive and a negative charge while being overall neutral.
The isoelectric point
As pH changes, the charge state changes. At low pH both groups are protonated and the molecule is cationic; at high pH both are deprotonated and it is anionic. Between them lies the pH at which the net charge is zero — the isoelectric point.
| Side chain | pI relative to neutral amino acids | Reason |
|---|---|---|
| Neutral | Around 6 | Average of the two ionisable groups |
| Acidic | Lower | An extra acidic group must also be protonated |
| Basic | Higher | An extra basic group must be deprotonated |
At its isoelectric point an amino acid does not migrate in an electric field, and its solubility is at a minimum — both because the net charge is zero. Those two facts are the basis of separation by electrophoresis and of isoelectric precipitation.
Titration curves
Titrating an amino acid from low pH gives a curve with two buffering regions, one for each ionisable group, and a steep section between them containing the isoelectric point. An amino acid with an ionisable side chain shows a third region.
Reading the pK values from the flat portions of the curve, and locating the isoelectric point midway between the relevant pair, is a standard exercise.
The peptide bond
Amino acids join by amide linkages formed with loss of water. The resulting peptide bond has properties that are not obvious from drawing it as a single bond.
- It is planar. The nitrogen lone pair delocalises onto the carbonyl oxygen, giving partial double bond character and restricting rotation.
- The trans arrangement is strongly preferred, because the cis form has a steric clash between adjacent side chains.
- The nitrogen is not basic in the way an ordinary amine is, since its lone pair is delocalised.
Planarity is what constrains protein folding: rotation is possible only about the two other backbone bonds, which is why protein conformation is described by just two angles per residue.
Determining sequence
A peptide's sequence is not determined by its composition, so it must be established separately.
| Method | Reveals |
|---|---|
| Complete hydrolysis | Composition only — which amino acids and how many |
| N-terminal labelling | Which residue is at the free amino end |
| Edman degradation | Removes and identifies one N-terminal residue at a time, leaving the rest intact |
| Partial hydrolysis | Overlapping fragments whose sequences can be pieced together |
Edman degradation is the important one because it is repeatable — each cycle removes one residue and leaves a shorter peptide that can be cycled again, so a sequence can be read off directly.
Frequently asked questions
Why do amino acids have such high melting points?
Because in the solid state they exist as zwitterions, so the lattice is held together by ionic forces rather than by the weaker forces typical of neutral organic molecules.
Why is solubility lowest at the isoelectric point?
Because the molecules carry no net charge there, so they do not repel one another and aggregate more readily.
Why is the peptide bond planar?
Because the nitrogen lone pair delocalises into the carbonyl, giving the C–N bond partial double bond character. Rotation about a partial double bond is restricted.
Why is the trans peptide bond preferred?
Because the cis arrangement places the side chains of adjacent residues close together, producing steric strain that the trans arrangement avoids.
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