Practical Protein Chemistry - A. Darbre 1989

Chemical Fragmentation of Polypeptides
Other Methods of Chemical Peptide Bond Cleavage
Cleavage of the Asn-Gly Bond

The property of hydroxylamine to specifically cleave the peptide bond of the Asn-Gly fragment (81) is successfully applied in practice for the structural studies of Proteins and large Peptides. The method is based on the ability of the asparagine side chain to cyclize, forming a substituted succinimide (82), which then undergoes nucleophilic

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attack by hydroxylamine [13]. The increased lability of the Asn-Gly bond compared to other Asn-X bonds is apparently due to the relative ease of cyclization in the absence of steric constraints from the side group of the neighboring amino acid residue (Glycine). It is known that certain glutamic acid esters in peptides also form cyclic compounds; however, no Cleavage of the Gln-Gly bond by hydroxylamine is observed.

The hydroxylamine cleavage Procedure is described in [i]. More recent publications focus mainly on various modifications of this technique, including increasing the hydroxylamine concentration, carrying out the reaction at a slightly elevated Temperature and a lower pH, using 6 M guanidine-HCl as a solvent, and employing lithium hydroxide to adjust the pH of the medium.

2.8.1.1. Procedure [44]. 102 mg of the factor X₁ light chain (as the S-pyridylethyl derivative) is dissolved at 20 °C in 25 ml of 6 M guanidine-HCl containing 2% hydroxylamine hydrochloride, and titrated with 4.5 M LiOH to pH 9.0. During the reaction, the pH is maintained using 4.5 M LiOH. The optimal reaction time is 4 h.