Practical Protein Chemistry - A. Darbre 1989

Polypeptide Fragmentation by Chemical Methods
Partial Acid Hydrolysis
Cleavage of the Asp-Pro Bond

The Asp-Pro peptide bond is particularly labile under mild acid Hydrolysis conditions [58, 73, 97, 108]. Selective Cleavage can occur in an acidic environment during Pepsin Hydrolysis, upon protein Treatment with a Cyanogen bromide solution in 70% formic acid, or during peptide purification by Gel filtration in aqueous acetic or formic acid. Partial hydrolysis of the Asp-Pro bond was observed, in particular, when Glutamate dehydrogenase was treated with cyanogen bromide in 70% formic acid [140]; the same study discusses the probable mechanism of acid hydrolysis of the Asp-Pro peptide bond.

Unfortunately, the Asp-Pro bond is relatively rare in Proteins; nevertheless, this approach is useful as it yields relatively large fragments suitable for automated sequencing.

2.3.1.1. Procedure [130]. The S-pyridylethylated derivative (obtained by reaction with 4-vinylpyridine) of the fetal Hemoglobin y-chain (51 mg) is dissolved in 2 mL of 70% formic acid containing 7 M guanidine-HCl and incubated at 40 °C for 24 h. Then, 2 mL of chilled deionized Water is added to the reaction mixture, and it is immediately chromatographed in 9% acetic acid on a Sephadex G-75 (superfine) Column (2X95 cm). The C-terminal fragment (residues 100–146) is obtained after lyophylization of the appropriate fractions followed by rechromatography on a Sephadex G-50 (superfine) column (2X95 cm), also in 9% formic acid.