Protein Hydrolysis: Acid And Alkaline Method

There are three general methods to hydrolyze protein into its composition, amino acids. Those methods are acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Strong acid is ordinarily the method of choice, and constant boiling hydrochloric acid, 6 M, is most frequently used. The reaction is usually carried out in evacuated sealed tubes or under N2 (Nitrogen) at110 Celcius degree for 18 to 96 hours. Under these conditions, peptide bonds are quantitatively hydrolyzed (although relatively long periods are required for the complete hydrolysis of bonds to valine, leucine, and isoleucine).


While the complete alkaline hydrolysis of proteins, is achieved with 2 to 4 M sodium hydroxide at 100 Celcius degree for 4 to 8 hours. This is of limited application for routine analysis, because cysteine, serine, threonine, and arginine are destroyed in the process, and partial destruction by deamination of other amino acids occurs. The complete enzymatic hydrolysis of proteins is difficult, because most enzymes attack only specific peptide bonds rapidly.

In this particular I only provide two methods of protein hydrolysis, acid hydrolysis and alkaline hydrolysis. Here are the methods:

Materials that you need:

3M p-toluenesulfonic acid.

0.2% tryptamine 3-[2-Aminoethyl] indole.

3M mercaptoethanesulfonic acid (Pierce).

1M sodium hydroxide.

Acid Hydrolysis of Protein:

1. 1 mL of 3M p-toluenesulphonic acid, containing 0.2% tryptamine (0.2% 3-[2-aminoethyl] indole) is added to the protein dried in a Pyrex glass tube (1.2 x 6 cm or similar, in which a constriction has been made by heating in an oxygen/gas flame).
2. The solution is sealed under vacuum and heated in an oven for 24 to 72 hours at 110 Celcius degree.
3. Altematively, you can use 3M mercaptoethanesulfonic acid as p-toluenesulphonic acid replacing, The sample is hydrolyzed for a similar time and temperature.
4. The tube is allowed to cool and cracked open with a heated glass rod held against a horizontal scratch made in the side of the tube.
5. The acid is taken to near neutrality by carefully adding 2 mililiters of 1M sodium hydroxide.
6. After this hydrolysis you can continue carrying out to quantitatively analyze certain amino acids, such as tryptophan.

Alkaline Hydrolysis Protein:

1. 0.5 mL of 3M sodium hydroxide is added to the protein dried in a Pyrex glass tube.
2. The solution is sealed under vacuum and heated in an oven for 4 to 8 hours at 100 Celcius degree.
3. After cooling and cracking open, the alkali is neutralized carefully with an equivalent amount of 1M HCl.