Hydrolysis: how water breaks peptide bonds

Hydrolysis means "splitting with water". In a hydrolysis reaction, a water molecule is used up to break a chemical bond, and its atoms end up on the two pieces. The peptide bonds that hold a peptide together can be broken this way. This guide explains how that happens, how fast, which parts of a sequence are most at risk, and how labs use hydrolysis on purpose to analyse peptides.

Terms are defined in the glossary.

What hydrolysis is

A peptide bond forms when the carboxyl group of one amino acid joins the amino group of the next and a molecule of water is released. Hydrolysis is the reverse: water is added back and the bond breaks. See what is a peptide for how the bond forms.

In the reaction, the oxygen of the water attacks the carbon of the peptide bond. The bond between that carbon and the nitrogen breaks. One piece ends with a free carboxylic acid (–COOH), and the other begins with a free amine (–NH₂).

Hydrolysis isn't special to peptides. The same kind of reaction splits esters, sugars joined in chains, and the phosphate bonds in molecules such as ATP and NAD+. See NAD+: structure and stability.

How fast it happens

The peptide bond is unusually stable. Its partial double-bond character makes the carbon less open to attack. Without a catalyst, at neutral pH and 25 °C, hydrolysis is very slow. Two well-known studies estimated the half-life of a typical peptide bond in water at about seven years and several hundred years respectively, using different methods (Radzicka & Wolfenden, 1996; Smith & Hansen, 1998).

That sounds reassuring, but a peptide has many bonds, and the rate rises sharply with:

• pH. Both strong acid and strong base speed up hydrolysis a great deal. The rate is lowest around neutral pH (Smith & Hansen, 1998).
• Temperature. As with most reactions, warmer means faster.
• Enzymes. Proteases cut peptide bonds in seconds that would otherwise last for years. Trace contamination with a protease can degrade a peptide in solution quickly.
• Water availability. Hydrolysis needs water. In a dry, freeze-dried powder there is very little, so the reaction is extremely slow. See what lyophilisation is.

Weak points in a sequence

Not every peptide bond is equally stable. Some sequences are known weak points:

• Aspartic acid (Asp, D). The side-chain carboxyl group can attack the neighbouring backbone bond, forming a ring. This makes bonds next to aspartic acid break more easily, especially under acidic conditions.
• Asp–Pro bonds. The bond between aspartic acid and proline is particularly easy to break in acid.
• Asp–Gly and Asn–Gly. These sequences readily form the same ring, which leads to deamidation and isomerisation as well as cleavage.
• The N-terminal two residues, especially when the second is proline or glycine. The free amine can attack the second peptide bond, cutting off the first two residues as a small ring called a diketopiperazine.

You can check a sequence for these motifs using the one-letter code. See amino acid codes.

How hydrolysis shows up in analysis

• On HPLC, the fragments usually appear as new peaks, often earlier than the main peak, because shorter pieces tend to be less hydrophobic. See how HPLC works.
• On mass spectrometry, each fragment has its own mass. The two fragments from one cut add up to the original mass plus 18.01 Da. A peak at a mass matching part of the sequence suggests the chain has been cut at that point. See reading impurity peaks.

Hydrolysis products look similar to truncated sequences left over from synthesis, which are also shorter pieces of the target. Comparing results over time helps tell them apart: synthesis by-products are there from the start, while hydrolysis products grow during storage. See shelf life and stability.

Limiting hydrolysis in storage

• Keep peptides dry. Store them freeze-dried rather than in solution where possible, sealed, with a desiccant. See hygroscopic powders.
• Keep them cold. Store at −20 °C or below for the long term. See how to store lyophilised peptides.
• Avoid extremes of pH in solutions, unless a method calls for them. See buffers and pH.
• Keep solutions clean to avoid contamination with proteases from microbes or skin.

Hydrolysis on purpose

Labs also use hydrolysis deliberately, to take a peptide apart and analyse the pieces.

Amino acid analysis

In amino acid analysis (AAA), the peptide is hydrolysed completely to its individual amino acids, classically in 6 M hydrochloric acid at about 110 °C for 24 hours, in a sealed tube with the air removed. The amino acids are then separated and measured. Comparing the amounts with those expected from the sequence confirms the composition and gives the peptide content of the sample.

Acid hydrolysis isn't perfectly clean. It destroys tryptophan, partly degrades cysteine, serine and threonine, and converts asparagine and glutamine into aspartic acid and glutamic acid. Labs correct for these losses or use special conditions. See NMR, FTIR, amino acid analysis and peptide mapping and net peptide content vs purity.

Enzymatic digestion and peptide mapping

Proteases cut only at particular residues. Trypsin, for example, cuts after lysine and arginine. Digesting a larger peptide or protein with a known protease and measuring the fragments by mass spectrometry confirms its sequence piece by piece. This is called peptide mapping.

Related guides

For other ways peptides change in storage, see shelf life and stability. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.