Peptide Cleavage, Separation and Analysis

To facilitate sequence analysis, the polypeptide chain is cleaved into fragments of manageable size reproducibly with enzymatic and/or chemical methods (Table 5.1). 

The common methods for determining N-terminal residue employ l-fluoro-2,4-dinitrobenzene (Sanger s reagent) and 1-dimethylamino phthalene-5-sulfonyl (dansyl) chloride. The derivatized peptide is hydrolyzed, and the labeled N-terminal residue is identified by its yellow color as dinitrophenyl (DNP) amino acid or by fluorescence as dansyl 

Procedures for determining the C-terminal residue of proteins involve hydrazenol-ysis and reduction with hydrides (directly or after esterification), though they are not widely used. The C-tenninal residue is identified as either the anuno acid or its alcohol after hydrolysis. In the hydrazenolysis, the peptidic hydrazide can be ronoved by extracting with benzaldehyde, which forms hydrazone with hydrazide. 

The peptide sequence determination can be preceded via stepwise (cyclic) degrada-tion/identification of N-terminal residues or C-terminal residues. This is accomplished by either chemical or enzymatic methods. 

An analogous Stark degradation releases the C-terminal residues successively as thiohydratoin (TH) amino acids (Bailey and Shively, 1990). 

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