Practical Protein Chemistry - A. Darbre 1989

Enzymatic fragmentation of the polypeptide chain
Protein modification methods
Modification of arginine residues

A number of Reagents have been proposed for the modification of Arginine residues in Proteins, such as malronic anhydride [53], cyclohexane-dione [105], 2,3-butanedione [121], and nitromalonaldehyde [95]. Information on certain modification Methods can be found in [120]. Unfortunately, in most cases the reaction is carried out under harsh conditions (at extreme pH levels), which leads to the irreversible modification of arginine residues and is accompanied by Side Reactions. Below is a Procedure using 1,2-cyclohexanedione [75]. The resulting arginine derivatives are stable at acidic pH and form a borate-ion complex that is stable at pH 8. The method is described in [98].

Modification of arginine residues with 1,2-cyclohexanedione. The protein is dissolved in 0.2 M Na-borate buffer (pH 9.0; 10 mg/mL) and incubated with 0.15 M 1,2-cyclohexanedione at 35 °C for 2 h. The reaction mixture is acidified with an equal volume of 30% acetic acid, the excess reagent is removed by dialysis against a dilute acetic acid solution, and the dialysate is lyophilized. According to an alternative protocol, upon completion of incubation, the reaction mixture is dialyzed against 0.1 M Na-borate buffer (pH 8.0). This yields a preparation suitable for Enzymatic Hydrolysis.

The modified arginine residues are unblocked by incubation at pH 7.0 in a 0.5 M hydroxylamine solution at 35 °C under an inert gas atmosphere for 12 h.