Practical Protein Chemistry - A. Darbre 1989

Chemical Fragmentation of Polypeptides
Partial Acid Hydrolysis
Side Reactions

Partial acid Hydrolysis of Proteins is accompanied by A number of side reactions, such as disulfide exchange, formation of diketopiperazines, cyclization of N-terminal glutamine to pyrrolidonecarboxylic acid, α–β-migration of aspartic acid residues, hydrolysis of asparagine and glutamine to yield carboxylic acids, and partial destruction of Tryptophan residues.

The disulfide exchange reaction [Equation (2.12)] occurs in strongly acidic media primarily via a mechanism involving The formation of sulfonium ions RS⁺ [156, 212]. In fact, the hydrolysis of Insulin in concentrated HCl yields an excess of cystine-containing Peptides (relative to the number of S—S bonds) [156]. Consequently, the disulfide exchange reaction during partial acid hydrolysis significantly complicates the localization of Disulfide Bonds.

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If dipeptides are formed during partial Protein Hydrolysis, they may not correspond to the native fragment due to the inversion of the Amino Acid Sequence. This inversion is caused by the cyclization of dipeptides into diketopiperazines, which readily occurs in dilute acids at room Temperature [Equation (2.13)]. Hydrolysis of these cyclic compounds can yield either the original or an isomeric dipeptide with a reversed amino acid sequence. Diketopiperazines are less stable under acid hydrolysis conditions compared to the corresponding dipeptides. The inversion of The amino acid sequence in 0.1 M HCl is clearly demonstrated using glycylvaline as an example [158].

The α,β-transpeptidation reaction of the aspartic acid residue proceeds in an acidic environment via The intermediate formation of a succinimide derivative [Equation (2.14)] [128, 185]. The resulting β-aspartyl derivative renders the peptide bond resistant to hydrolysis by exopeptidases, such as leucine aminopeptidase and Carboxypeptidases. Furthermore, the β-aspartyl imide blocks Edman sequencing at the cyclization stage.

Under relatively mild conditions, the N-terminal glutamine residue (18) undergoes cyclization to form pyrrolidonecarboxylic acid (19)

[77, 177]. Therefore, peptides with an N-terminal glutamine give a negative ninhydrin reaction and cannot be sequenced. Glutamic acid residues can undergo a similar cyclization, albeit under harsher conditions. Regarding the Cleavage of N-terminal pyrrolidonecarboxylic acid, see Section 1.6.1.