Practical Protein Chemistry - A. Darbre 1989

Determination of the composition of protein oligomers. Preparation of monomers and polypeptide chains
Preparation of monomers for sequencing
Hydrolysis of N-terminal pyroglutamic acid

Ideally, when sequencing well-prepared samples via Edman Degradation, the yield of the N-terminal amino acid should be quantitative. In other words, the samples must be free of salts and impurities, and the N-terminal amino acid should be devoid of protecting groups. During purification, any modification of amino acid functional groups should be avoided, as automated sequencing requires a maximal yield at each cycle. Precautions to prevent the blocking of N-terminal Amino Acids and The formation of heterogeneous preparations were briefly discussed in previous sections. This section covers specialized protocols for preparing Proteins for analysis, including Methods for delipidation and the Cleavage of oligosaccharide chains in carbohydrate-rich Glycoproteins.

The Development of an efficient method for cleaving off the pyroglutamic acid residue resolved the sequencing challenge for proteins blocked by the cyclization of N-terminal Gln and Glu [136]. Because this high-yield 'deblocking reaction' exposes the α-amino group of the subsequent amino acid residue, the cyclization reaction can actually be viewed as a convenient way to introduce a protective group during Protein Purification stages. Deprotection is carried out by double incubation with bovine Liver pyroglutamate aminopeptidase. This method was first applied to the analysis of immunoglobulin light and heavy chains, and subsequently to the heavy chain of the Influenza virus hemagglutinin [181].

1.6.1.1. Cleavage of the N-terminal pyroglutamic acid residue [136]. Bovine liver pyroglutamate aminopeptidase (L-pyroglutamyl-peptide hydrolase, EC 3.4.11.8) is stored lyophilized at −20 °C. A solution (10 mL, 1 mg/mL) of the reduced and alkylated protein is dialyzed at 4 °C against 0.1 M phosphate buffer (pH 8) containing 5 mM DTT + 10 mM EDTA + 5% (v/v) glycerol. The preparation is then transferred to a reaction vial with a tightly sealed cap, 0.025 mg of the enzyme is added, flushed with nitrogen, tightly capped, and mixed. Incubation is carried out at 4 °C for 9 h with occasional mixing. A second portion of the enzyme (0.025 mg) is added, and the mixture is incubated again under a nitrogen atmosphere at 20 °C for 14 h. The reaction mixture is dialyzed against 0.05 M acetic acid, lyophilized, and the protein sample is subjected to automated sequencing.

A method has been developed for the Hydrolysis of pyroglutamic acid yielding N_y-methylglutamine [125]. The protected peptide is incubated in a 17% methylamine solution at 37 °C for 14 h (or longer). Following Dansylation and subsequent hydrolysis, DNS-glutamic acid is identified.