Practical Protein Chemistry - A. Darbre 1989

Disulphide bonds
Cleavage of the polypeptide chain into short cystine-containing peptides

As noted earlier, protein fragmentation should be carried out under conditions that prevent disulfide exchange, preferably within the pH range of 2–6.5. Nevertheless, to achieve satisfactory results, it is often necessary to conduct the reaction at more alkaline pH values. For instance, Digestion with Trypsin and Thermolysin at pH 7.0–7.5 has been successfully employed [19, 30, 41, 42]. However, given the varying stability of Disulfide Bonds in Proteins, one should avoid drawing hasty Conclusions when interpreting the results obtained under such conditions.

Pepsin is frequently used for initial protein Processing due to its high activity in acidic environments and its ability to hydrolyze native proteins. Once the stable Structure OF THE native protein has been disrupted by pepsin, subsequent digestion with other Enzymes can be performed at a pH optimal for preserving disulfide bonds. Cyanogen bromide Cleavage at Methionine Residues in a weakly acidic medium is widely used to determine the positions of disulfide bonds. This method offers the advantage of yielding well-defined fragments that can be isolated and utilized for subsequent fragmentation.

The choice of enzymes for subsequent cleavage is dictated by various factors and aims to produce small Peptides containing a single disulfide bond. Treatment with trypsin, Chymotrypsin, staphylococcal protease, or thermolysin—either individually or in various combinations—is most commonly employed. Occasionally, enzymes with narrower Specificity, such as plasmin or Elastase, are successfully used [5, 11]. For the analysis of peptides containing two disulfide bonds separated by a few amino acid residues, Edman Degradation is applied [20].

The Specific features of cleavage Methods used for disulfide bond determination are discussed in this chapter; for protease Hydrolysis, see Chapters 3 and 10, and for chemical cleavage methods, see Chapters 1 and 2.