Peptide bond polarity: Like all amide linkages, the −C = O and −NH groups of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The −C = O and −NH groups of the peptide bond are polar, however, and are involved in hydrogen bonds (for example, in α-helices and β-sheets), as described on pp. 16–17.
B. Determining the amino acid composition of a polypeptide
The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the attached group is positively charged, the column becomes an anion-exchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH (Fig. 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin (a reagent that forms a purple compound with most amino acids, ammonia, and amines). The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer, an automated machine whose components are depicted in Figure 2.3.
C. Sequencing the peptide from its N-terminal end
Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Fig. 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond such that it can be hydrolyzed without cleaving the other peptide bonds. The identity of the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle. Automated sequencers are now used.
D. Cleaving the polypeptide into smaller fragments
Many polypeptides have a primary structure composed of >100 amino acids. Such molecules cannot be sequenced directly from end to end. However, these large molecules can be cleaved at specific sites and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thereby providing a complete amino acid sequence of the large polypeptide (Fig. 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.]
E. Determining a protein’s primary structure by DNA sequencing