Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Protein modification methods
Alkylation of sulfhydryl groups

Several alkylating agents are known, and the choice of reagent depends on The properties of the functional group to be introduced into the polypeptide chain. The three most common alkylation Methods are discussed below.

3.4.2.1. Carboxymethylation. Carboxymethylation is the most widely accepted method for modifying protein sulfhydryl groups. Iodoacetic acid or iodoacetamide are used as Reagents to introduce acidic or neutral groups. Both reagents are recrystallized beforehand to remove free iodine: iodoacetic acid from hexane, and iodoacetamide from petroleum ether. Both reagents are added to the reaction mixture in slight excess relative to the total concentration of sulfhydryl groups. The iodoacetic acid solution is neutralized with Tris or NaOH before use. The reaction is carried out at 0 °C for 1 h. Under these conditions, the alkylation of other protein functional groups is prevented. The method is discussed in detail in reference [37] and in Section 2.2.1.

Both reagents are commercially available as radiolabeled preparations and can be used to introduce a radioactive label into the polypeptide chain. When introducing a label, the radioactive preparation (iodoacetic acid or iodoacetamide) is added first, followed by a slight excess of the "cold" reagent. This achieves the highest degree of label incorporation, since the reactivity of sulfhydryl groups is known to be significantly higher than that of the reducing agent present in the reaction mixture.

3.4.2.2. Aminoethylation. The S-aminoethyl group is introduced into the polypeptide chain by alkylating sulfhydryl groups with ethyleneimine [81, 82]. This type of modification is of great importance because aminoethylcysteine is a Lysine analog that is sensitive to Trypsin action (Section 3.5.1). It has been shown that an eightfold excess of ethyleneimine per sulfhydryl group must be used [12]. At low pH values, a single side reaction involving the alkylation of Methionine residues is observed [91]. Therefore, acidification of the reaction mixture should be avoided until the alkylating reagent is completely removed. The method is discussed in more detail in Chapter 2.

3.4.2.3. Other alkylation reactions. 4-Vinylpyridine is used to obtain the acid-stable 5-pyridylethylcysteine residue [26]. The Use of 2-bromoethyltrimethylammonium bromide as an alkylating reagent yields a Cysteine derivative that is also stable under acidic Hydrolysis conditions and automated sequencing conditions [45]. The method is discussed in more detail in Chapter 2.