Practical Protein Chemistry - A. Darbre 1989

Disulfide Bonds
Cleavage of the polypeptide chain into short cystine-containing peptides
Hydrolysis following maleylation and succinylation

There are instances where a protein retains its resistance to proteases even after Denaturation with 8 M urea, 6 M guanidine-HCl, boiling, or exposure to an acidic environment. An approach for analyzing such Proteins was developed in [5]. Although toxin B-IV (a crustacean-selective neurotoxin) proved to be extremely resistant to proteolysis in both its native and denatured states, its derivatives (maleyl and succinyl) were sensitive to Trypsin.

4.4.6.1. Succinylation [9]. To a 1% protein solution in 1.0 M Na-bicarbonate buffer (pH 8.0) at 25 °C, 0.8 parts (by weight) of succinic anhydride are gradually added over 1 h. The pH of the reaction mixture is maintained at 8.0 by adding 0.1 M NaOH. After 1–2 h, the reaction mixture is diluted with 2.5 volumes of distilled Water and dialyzed for 12 h. The modification of certain proteins can quite feasibly be carried out under milder conditions [22]. The modified protein can then be incubated simultaneously in the presence of trypsin, Chymotrypsin, and Thermolysin at an Enzyme-to-substrate ratio of 1 : 50. Incubation conditions: 0.1 M K-phosphate buffer (pH 6.8), 40 °C. After 4 h, the solution is acidified to pH 3.0 and lyophilized.

4.4.6.2. Maleylation [5]. Partial maleylation can render a proteolysis-resistant protein sensitive to trypsin action. The reaction is carried out using a 10-fold excess of maleic anhydride (per amino group) at pH 8.0 for 5 h. The pH of the mixture is then adjusted to 7.0, and the modified protein is incubated with trypsin at 37 °C at an enzyme-to-substrate ratio of 1 : 33. The reaction is stopped by acidification to pH 3.0. Removal of the protecting groups is performed at pH 3.5 and 37 °C for 48 h [7]. The peptide mixture can be subjected to further Hydrolysis by trypsin or other proteases.

It should be noted that during modification and hydrolysis, the protein is kept in a neutral or slightly alkaline environment, i.e., under conditions that do not, in principle, rule out disulfide exchange. Regarding maleylation, see Section 3.4.2.2.