Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Hydrolysis of Proteins and Peptides
Sample Preparation for Hydrolysis
Hydrolysis of Proteins and Peptides in 6 N HCl

Principle of the method. Hydrolysis is carried out at 105°C in 6 N HCl in a sealed ampoule.

Scope of application. Hydrolysis of Proteins AND Peptides to determine their Amino Acid Composition.

PROCEDURE

a) Purification of HCl. Concentrated HCl is repeatedly distilled in a Glass distillation apparatus (Quickfit, USA) over SnCl₂. The constant-boiling fraction is used in the experiments.

b) Hydrolysis. The material to be hydrolyzed is dissolved or suspended in a 100- to 200-fold excess of 6 N HCl in an ampoule.

The ampoule is flushed with nitrogen or evacuated, then sealed and incubated at 105°C for 24–72 h.

c) Removal of Hydrochloric acid. Large-volume hydrolysate is diluted with distilled Water and then evaporated in a rotary evaporator. The dry residue is suspended in a small amount of distilled water, and the evaporation is repeated. A small volume of the hydrolysate is transferred to a Petri dish and placed in a vacuum desiccator over KOH or P₂O₅ to remove the acid.

NOTE

Some Amino Acids are destroyed during hydrolysis: Tryptophan is completely decomposed, carboxymethylcysteine by 50–60%, and Threonine and Serine by 5–10%. These losses should be taken into account during quantitative analysis by introducing appropriate correction factors. The faster the acid is removed, the lower the likelihood of undesirable Side Reactions.