Hydrolysis of Proteins

Hydrolysis of proteins can be carried out by the use of strong acid, strong base or proteolytic enzymes as catalysts. However, acid hydrolysis is the usual procedure.

Acidic Hydrolysis:

The acidic hydrolysis of proteins is generally carried out in evacuated sealed tubes at about 110°C for periods ranging from 12 to 96 hours. Under these conditions, the peptide bonds are quantitatively hydrolysed to yield the amino acid hydrochlorides. Undesirable side-effects of acidic hydrolysis are as follows:

A. Among the amino acids that usually occur in proteins, all the tryptophan and variable amounts of cysteine and cystine are slowly destroyed. Serine and threonine are slowly destroyed. Formation of humin, a black polymer of breakdown product of tryptophan, acccompanies acid hydrolysis.

B. Glutamic and asparagine are deamidated to glutamate and aspartate.

C. Glutamic acid undergoes intramolecular dehydration to pyrrolidone 5-carboxylic acid.

D. Other amino acids may undergo intermolecular dehydration forming cyclic anhydrides or diketopiperazines.

In spite of these difficulties, acidic hydrolysis is ordinarily the method of choice because the amino acids are not racemised by acid hydrolysis.

Alkaline Hydrolysis:

The complete hydrolysis of proteins may also be carried out by incubating them with 2 to 4N sodium hydroxide or 5N barium hydroxide at 100°C for 4 to 8 hours. Such hydrolysis is of limited use due to the following difficulties:
A. Cysteine, cystine, serine, threonine and arginine are destroyed in this process.
B. Other amino acids may be partially destroyed by deamination.
C. Racemisation of amino acids occurs.

In spite of the above mentioned difficulties, the alkaline hydrolysis is useful for the analysis of tryptophan because it is not destroyed during alkaline hydrolysis.

Enzymatic Hydrolysis:

This technique is very valuable for the elucidation of amino acid sequence in a protein. However, the complete enzymatic hydrolysis is slow and difficult because most enzymes attack only specific types of linkages rapidly. Often, by use of a number of enzymes each in turn, it is possible to degrade the protein into smaller and smaller fragments and ultimately into the constituent amino acids.

The bacterial peptidases subtilisin and pronase catalyse hydrolysis of all peptide bonds but the reaction is slow compared to acid catalysed hydrolysis. Other proteolytic enzymes (trypsin, chymotrypsin), catalyse hydrolysis of certain peptide bonds quite rapidly. The enzymatic hydrolysis is complicated, owing to possible contamination resulting from proteolysis of the enzymes themselves.

Source: Biochemistry by C.B. POWAR and G.R. CHATWAL