Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Proteases with low specificity
Chymotrypsin

This section provides information on practically important Enzymes (whose properties are listed in Table 3.2) with a broader Specificity. While not exhaustive, it covers the majority of proteases with broad specificity. Many of these hydrolyze peptide bonds adjacent to hydrophobic amino acid residues. Enzymes such as Pepsin and Papain are also discussed, as they are frequently used for the Limited proteolysis of native Proteins.

Chymotrypsin is one of the most specific enzymes discussed in this section; it is widely used to obtain primary hydrolysates [50, 56, 99].

Specificity. Chymotrypsin predominantly hydrolyzes the C-terminal peptide bond of an aromatic or bulky hydrophobic residue of the -H-X- type, where X = Tyr, Phe, Trp, or Leu. Bonds of the -H-Pro- type are resistant to the enzyme's action. Chymotrypsin exhibits a broader specificity compared to Trypsin, as it can cleave peptide bonds of other amino acid residues, such as Leu, His, Ile, Met, Ser, and Val.

Hydrolysis conditions. Hydrolysis is carried out under conditions similar to those for trypsin Digestion: in 100 mM NH₄HCO₃, Enzyme-to-substrate ratio = 1 : 50, at 37 °C for 4 h. Occasionally, a pH 7.0 buffer containing 2 M guanidine-HCl is used [101]. Chymotrypsin is a Serine protease and is inhibited by DFP and PMSF. Trypsin contamination in chymotrypsin preparations is rare. If necessary, any trypsin activity can be eliminated by adding soybean trypsin inhibitor to the reaction mixture (Section 3.5.1.1).