Chemistry and Biology of Proteins - F. Haurowitz 1953

Hydrolytic cleavage of proteins
Methods of hydrolysis

Protein Hydrolysis is typically carried out using acids, alkalis, or appropriate Enzymes. In most cases, boiling Hydrochloric acid is used for hydrolysis (commercial 35% hydrochloric acid is diluted to 20.5% before use). The dry protein is mixed with approximately a 10-fold volume of 20.5% acid and placed in a boiling Water bath for 30–60 min to prevent vigorous foaming. The mixture is then refluxed for 12–48 h. Upon completion of hydrolysis, most of the hydrochloric acid can be removed from the hydrolysate by conventional distillation or, preferably, vacuum distillation. Free Amino Acids are isolated from the dry residue, which contains amino acid hydrochlorides, by Treatment with moist silver oxide. However, this Procedure may cause undesirable Side Reactions, specifically The formation of complex salts with silver by Certain amino acids. Therefore, if the isolation of free amino acids from the hydrolysate is intended, it is more advantageous to substitute sulfuric acid for hydrochloric acid. For protein hydrolysis, 8 N H₂SO₄ is used (20 mL per 100 mL of solution). Sulfuric acid is removed from the hydrolysate by adding an equivalent amount of barium hydroxide. The heavy barium sulfate precipitate must be extracted several times with boiling water to recover significant amounts of amino acids adsorbed on the precipitate.

Acid Hydrolysis of Proteins does not lead to Amino Acid Racemization, meaning the amino acids are obtained as l-amino acids. This is the main advantage of acid hydrolysis over alkaline hydrolysis. Most amino acids are stable against boiling mineral acids. Exceptions are Tryptophan, which is completely destroyed during such hydrolysis, and the hydroxy amino acids Serine and Threonine, which are partially destroyed. Tryptophan degradation products are converted into dark brown substances called humins; humins are likely formed through the Condensation of the tryptophan indole ring with small amounts of aldehydes generated during hydrolysis [1]. The formation of humins can be prevented by adding tin to the sulfuric acid used for hydrolysis. Due to the reducing action of tin, this addition prevents The conversion of Cysteine into cystine [2], but it does not protect tryptophan from destruction. The same goal can be achieved by carrying out hydrolysis in a mixture of 20% hydrochloric and 50% formic acids [3].

The destruction of tryptophan can be avoided if protein hydrolysis is performed not with acids, but with boiling dilute caustic alkali or baryta. Because these bases exhibit very strong hydrolytic activity, complete protein hydrolysis with a 4 N barium hydroxide solution is achieved in 10 h [4]. Alkaline hydrolysates are colorless and contain no humins. A drawback of alkaline treatment is that it causes amino acid racemization. In addition, boiling with alkalis leads to the deamination of certain amino acids, the Cleavage of Arginine into Ornithine and urea, and the destruction of cystine and cysteine.

Protein hydrolysis can also be carried out using Proteolytic Enzymes. Although Enzymatic Protein Hydrolysis proceeds under very mild conditions, it is rarely used for preparative purposes because complete hydrolysis requires a very long time. Another drawback of this method is that the hydrolysates become contaminated with the degradation products of the enzyme itself, which is also a protein.

To gain a more precise understanding of Cell/13.html">Protein Structure, partial protein hydrolysis is frequently employed. Partial hydrolysis can be achieved in several ways: 1) by reducing the hydrolysis time; 2) by lowering the concentration of the hydrolyzing agent; 3) by conducting the hydrolysis at a reduced Temperature [5].

Thus, for example, the hydrolysis of globin with 25 N sulfuric acid at 40° yielded a Histidine peptide [6]. Similarly, dipeptides were isolated from hydrolysates obtained by treating proteins with concentrated hydrochloric acid at 37° [7]. The formation of Peptides during hydrolysis indicates that various peptide bonds differ in their resistance to the hydrolyzing agent. If, for instance, egg albumin is treated with 23% hydrochloric acid at temperatures ranging from 30 to 60°, only 56 peptide bonds of this protein are rapidly cleaved [8].

Enzymes can also be used for the partial hydrolysis of proteins. This approach allows the isolation of asparagine [9] and glutamine [10] from edestin and gliadin, respectively. During conventional complete hydrolysis, both of these amides are hydrolyzed to yield aspartic and glutamic acids and ammonia. Since individual proteolytic enzymes hydrolyze only specific peptide bonds, the action of different enzymes yields mixtures of various peptides. For example, peptides containing N-linked Proline are cleaved by erepsin but are not cleaved by Trypsin [11].