Sanger degradation

Sanger degradation Peptide sequencing by tagging succe.ssive /V-terminal amino acids—residues at the amino end of the chain. 

P-pleated sheet (p. 1191) primary structure (p. 1190) quaternary structure (p. 1194) random coil (p. 1191) ribonucleic acid (RNA) (p. 1211) Sanger degradation (p. 1196) secondary structure (p. 1191) side chain (p. 1175)... 

Sanger degradation (Section 23.4) A method for determining the amino acid at the amino terminus of a chain using 2,4-dinitrofluorobenzene. Unfortunately, the Sanger degradation hydrolyzes, and thus destroys, the entire peptide. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

FIGURE 3-25 Steps in sequencing a polypeptide, (a) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger s method for identifying the amino-terminal residue is shown here, (b) The Edman degradation procedure reveals... 

By the primary structure of a peptide or protein, we mean its amino acid sequence. Complete hydrolysis gives the amino acid content. The N-terminal amino acid can be identified by the Sanger method, using 2,4-dinitrofluorobenzene. The Edman degradation uses phenyl isothiocyanate to clip off one amino acid at a time from the N-terminus. Other reagents selectively cleave peptide chains at certain amino acid links. A... 

Know the meaning of amino acid sequence, Sanger s reagent, Edman degradation, selective peptide cleavage. 

Understand how peptides are sequenced, using Sanger s reagent, the Edman degradation, and enzyme hydrolysis. (Problems 26.37 and 26.38)... 

The thin-layer gel system used for resolving the different fragments produced by the chemical degradation method is essentially that of Sanger, Nicklen and Coulson (1977) as described in Chapter 4. An 8% polyacrylamide gel is usually used though for special purposes a 10 or 12% gel can give an improved resolution of the smallest fragments. For the standard 8% gel the polymerization mixture contains 7.6% (wt/vol) acrylamide, 0.4% (wt/vol) bisacrylamide, 50% (wt/vol) urea, 100 mM Tris-borate, pH 8.3, 2mM EDTA, 0.07% (wt/vol) ammonium persulphate and TEMED catalyst. This solution is poured or injected into a 0.4 x 200 x 400 mm mold to form a gel slab. 

The Sanger method for N-terminus determination is a less common alternative to the Edman degradation. In the Sanger method, the peptide is treated with the Sanger reagent, 2,4-dinitrofluorobenzene, and then hydrolyzed by reaction with 6 M aqueous HC1. The N-terminal amino acid is recovered as its 2,4-dinitrophenyl derivative and identified. 

The structure and properties of peptides and proteins depend critically upon the sequence of amino acids in the peptide chain. The first complete amino acid sequence of a protein, that of insulin (51 amino acid residues), was determined by F. Sanger in 1953. The process is now performed using automated protein sequencers, and involves step-by-step identification of amino acids at the N terminus of the protein using a chemical process known as Edman degradation. 

A discussion of much of the older literature on hydrolysis can be obtained from the reviews of Vickery and Osborne (1928), Synge (1943), and Green-stein and Winitz (1961). Reviews of more recent aspects of hydrolytic degradation are those of Sanger (1952), Leach (1953), Desnuelle (1953), Thompson (1960), and Light and Smith (1963). Methods for nonen-zymatic degradation have been discussed by Witkop (1961). 

Alkalies have not been used extensively for degradation of proteins and polypeptides. Their limitations have been recognized for some time, and few attempts have been made in recent years to further evaluate this medium for hydrolysis of proteins. Much of the available information on alkaline hydrolysis has been reviewed elsewhere (Sanger, 1952 Leach, 1953 Desnulle, 1953), and only a few comments are necessary here. 

The Sanger end-group determination is sometimes used as an alternative to the Edman degradation. In the Sanger method, a peptide is allowed to react witi> 2.4-dinitrolluorobenr ae. the peptide is hydrolyzed, and the N-terminal amino add is identified by separation as its iV-2,4-dinitrophenyl derivative. Propose a mechanism to account for the initial reaction between peptide and dinitrofluorobenzene. 

Direct sequencing techniques involve a variety of synthesis, degradation, or separation techniques, and include the traditional Sanger [ 1 ], pyrosequencing [2,... 

It is always useful to determine the N-terminal amino acid in the peptide even before the sequencing by Edman degradation is attempted. The N-terminal amino acid is determined by reacting the peptide with Sanger s reagent fluorodinitrobenzene (FDNB), and then the amino acids... 

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