Harper's Illustrated Biochemistry, Volume 1 - Murray R. 1993

Structure and Function of Proteins and Enzymes
Peptides
Determination of the Primary Structure of Polypeptides

More than three decades have passed since Sanger startled the biochemical world by determining the complete Introduction/19.html">Primary Structure of the hormone Insulin using chemical and enzymatic Methods. Sanger's approach was as follows. First, he separated the two polypeptide chains of insulin, A and B, and then carried out their specific enzymatic Cleavage into small Peptides containing overlapping sequence segments. He separated these peptides and identified their N-terminal residues using the 1-fluoro-2,4-dinitrobenzene reagent (Fig. 4.9). In addition, he determined the Amino Acid Composition of the peptides, which ultimately enabled him to establish their structure. By comparing the sequences of the overlapping peptides, he unequivocally established the primary structure of both chains, A and B.

In broad outline, Sanger's strategy has retained its significance to this day; however, over the ensuing decades, two new approaches have been developed that revolutionized the Determination of the primary structure of Polypeptides (Proteins). The first approach is based on the automated Procedure developed by Edman in 1967 for the sequential cleavage and identification of N-terminal amino acid residues as their phenylthiohydantoin derivatives. The second approach is associated with the method developed by Sanger and, independently, by Maxam and Gilbert, which allows for the rapid and unambiguous sequencing of the Gene encoding the protein in question. The optimal strategy consists in using both approaches simultaneously. Automated Edman Degradation, although much faster than Sanger's original manual method, is nevertheless vastly inferior in speed to DNA Sequencing Methods and is associated with A number of difficulties. On the other hand, DNA sequencing does not always yield the unambiguous primary STRUCTURE OF THE protein under investigation. At the same time, a major advantage of the DNA sequencing method is the relative ease of detecting and sequencing segments that are present only in the protein precursor and are cleaved off during its maturation. Thus, DNA sequencing and the automated Edman procedure Complement each other; their combined use has fundamentally transformed and expanded our knowledge of the Primary Structure of Proteins. We will discuss DNA sequencing in Chapter 38, and the Edman procedure in the next section.

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Fig. 4.9. Reaction of an amino acid with 1-fluoro-2,4-dinitrobenzene (Sanger's reagent). The reagent is named after Nobel laureate (1958) biochemist Frederick Sanger, who used it to determine the primary structure of insulin. Initially, all free Amino groups are arylated (with quantitative yield); following peptide Hydrolysis, brightly colored 2,4-dinitrophenyl Amino Acid Derivatives are formed. Quantitative analysis of these derivatives is carried out by Spectrophotometric Methods. Dinitrofluorobenzene also reacts with the ε-amino group of Lysine, the imidazole group of Histidine, the OH group of Tyrosine, and the SH group of Cysteine. Dinitrophenyl groups are not cleaved during acid hydrolysis and are used to identify N-terminal Amino Acids in polypeptides.