Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Preparation of the substrate

The completeness of Enzymatic Hydrolysis depends heavily on the physical state of the substrate. For instance, if a native protein undergoes proteolysis, the resulting hydrolysis is likely to be limited. However, if the goal is to obtain protein fragments with partial biological activity, this method is quite applicable. Such an approach is discussed in detail in the section dedicated to The Use of Trypsin, Pepsin, and Papain. In general, to ensure complete hydrolysis, the protein is denatured with the obligatory reduction of Disulfide Bonds.

In most cases, boiling for 5–10 min at a neutral pH or precipitation with a 5% trichloroacetic acid solution is sufficient. Disulfide reduction is carried out in the presence of a denaturing agent, such as 8 M urea or 6 M guanidine-HCl, followed by alkylation of the resulting Cysteine residues. Methods for disulfide reduction and subsequent alkylation are covered in Section 3.3 and Chapter 2.

Upon Denaturation, Protein solubility drops sharply; and while enzymatic hydrolysis of insoluble substrates is not a major issue in itself, handling such substrates can sometimes be challenging. To achieve successful enzymatic hydrolysis of an insoluble protein, the resulting precipitate should be converted into a fine suspension whenever possible. To this end, the protein is brought into solution at an extreme pH and then titrated to the required pH. Alternatively, the protein sample can be dissolved in 6 M guanidine-HCl, which is subsequently removed by dialysis. If these treatments do not yield satisfactory results, the protein is solubilized via reversible Modification of Lysine residues, such as citraconylation. As a result, the protein acquires an additional charge, which frequently leads to disaggregation.

Certain Proteolytic Enzymes partially or fully retain their activity under denaturing conditions and, consequently, can be used at low concentrations of urea, guanidine-HCl, or sodium dodecyl sulfate (SDS). In some cases, to enhance solubility, the buffer concentration is increased, or hydrolysis is performed at elevated temperatures. Detailed information is provided below on enzymes that maintain enzymatic activity under denaturing conditions or at elevated temperatures, with a special focus on trypsin, Clostripain, S. aureus V8 protease, Chymotrypsin, Thermolysin, and papain.