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

Hydrolysis of proteins and peptides
Tryptic hydrolysis of proteins and peptides

Principle of the method. Trypsin cleaves peptide bonds formed by Lysine and Arginine residues in a protein or peptide.

Applications. Cell/13.html">Protein Structure analysis.

PROCEDURE

1. Selection and pre-purification of a suitable trypsin preparation. Trypsin is the most thoroughly studied highly specific proteolytic enzyme capable of selectively cleaving peptide bonds formed by lysine and arginine residues. However, commercially available trypsin preparations derived from pancreatic extracts may be contaminated with other proteases; therefore, a trypsin preparation suitable for specific purposes must be carefully selected. It is best to use a preparation classified as “Chymotrypsin-free”. If such a preparation is unavailable, it is recommended to purify the available trypsin as follows: the enzyme is dissolved in 0.01 N HCl and incubated at 37°C for 16 h, then the solution is filtered and the pH is adjusted to 7.8.

2. Tryptic Hydrolysis. The material to be hydrolyzed is dissolved or suspended in a 0.1 M ammonium bicarbonate solution to a final concentration of 1% and ¹/₃₀ part of trypsin (relative to The amount of protein) is added. The mixture is incubated at 37°C for 3 h, then another ¹/₃₀ part of trypsin is added, incubated again for 1 h, the hydrolysate is placed in a boiling Water bath for 15 min, filtered, and lyophilized.

NOTES

1. Hydrolysis of insoluble Proteins and protein derivatives is recommended to be carried out in an autotitrator. The material to be hydrolyzed is suspended in distilled water, the pH is adjusted to 7.5 with a 0.05 N NaOH solution, trypsin is added, and the reaction is carried out at 37°C under a nitrogen atmosphere with constant stirring of the suspension and maintaining pH 7.5 by adding 0.05 N NaOH solution. Hydrolysis continues for 4–5 h.

2. Native proteins are either not attacked by trypsin at all or are digested extremely slowly. It should also be noted that tryptic hydrolysis can yield high-molecular-weight insoluble structures resistant to the enzyme, the so-called “core” structures.

3. By reversible or irreversible blocking of the ε-NH₂ groups of lysine residues, tryptic hydrolysis can be restricted to the Cleavage of arginine peptide bonds only. Reversible blocking can be achieved, for example, by trifluoroacetylation.