L-Fluoro-2,4-dinitrobenzene Sanger

Number of Polypeptide Chains in a Multisubunit Protein A sample (660 mg) of an oligomeric protein of Mr 132,000 was treated with an excess of l-fluoro-2,4-dinitrobenzene (Sanger s reagent) under slightly alkaline conditions until the chemical reaction was complete. The peptide bonds of the protein were then completely hydrolyzed by heating it with concentrated HC1. The hydrolysate was found to contain 5.5 mg of the following compound ... 

In this procedure (Fig. 11.2) all the amino groups of POase are first irreversibly blocked with l-fluoro-2,4-dinitrobenzene ( Sanger s reagent ). The carbohydrates of this enzyme (21% of total weight. 

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... 

N-terminal amino acids can be determined by treating a protein with l-fluoro-2,4-dinitrobenzene Sanger s reagent cf. 1.2.4.2.2) or 5-dimethy laminonaphthalene-1 - sulfony 1 chlor-... 

Arylation with l-fluoro-2,4-dinitrobenzene (Sanger s reagent FDNB) and trinitrobenzene sulfonic acid was outlined in Section 1.2.4.2.2. FDNB also reacts with cysteine, histidine and tyrosine. 

Several chemical methods have been devised for identifying the N-tenninal anino acid. They all take advantage of the fact that the N-tenninal anino group is free and can act as a nucleophile. The a-anino groups of all the other anino acids are part of amide linkages, are not free, and are much less nucleophilic. Sanger s method for N-tenninal residue analysis involves treating a peptide with l-fluoro-2,4-dinitrobenzene, which is very reactive toward nucleophilic aromatic substitution (Chapter 23). 

When Sanger s method for N-tenninal residue analysis was discussed, you may have wondered why it was not done sequentially. Simply start at the N terminus and work steadily back to the C terminus identifying one anino acid after another. The idea is fine, but it just doesn t work well in practice, at least with l-fluoro-2,4-dinitrobenzene. 

Sanger s reagent (Section 27.11) Thecompound l-fluoro-2,4-dinitrobenzene, used in N-terminal amino acid identification. 

Buyuktimkin and Buyuktimkin [20] described a spectrophotometric method of assay of penicillamine and its tablets. An aqueous solution of 100 mg/mL of penicillamine was added to ethanolic 5 mM Sanger reagent (l-fluoro-2,4-dinitrobenzene), solid NaHC03, and water. The solution was diluted and heated at 70 °C for 45 min. After cooling, the solution was diluted with 3% ethanolic HC1. The absorbance of the resulting yellow complex was measured at 355 nm (molar absorptivity = 19,721). Recovery of the drug from commercial tablets was 99.1 0.7%. 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

Carboxypeptidase-catalyzed hydrolysis can be used to identify the C-terminal amino acid. The N terminus is determined by chemical means. One reagent used for this purpose is Sanger s reagent, l-fluoro-2,4-dinitrobenzene (see Figure 27.9). 

Sanger s reagent, l-fluoro-2,4-dinitrobenzene (FDNB), which was used in the earlier days for the quantitation of primary amino groups by colorimetric determination, can also be used in the identification of amino-terminal residue, but not for sequencing. At the present time, N-terminal analysis is performed on a protein sequencer. 

Several methods are available to determine the N-terminal amino acid. In Sanger s method, the polypeptide chain is reacted with l-fluoro-2,4-dinitrobenzene. The dinitrophenyl (DNP) derivative of the N-terminal amino acid can be isolated and identified by ion-exchange chromatography after the polypeptide is hydrolyzed. A group of enzymes called the carboxypeptidases are used to identify the C-terminal residue. Carboxypeptidases A and B, both secreted by the pancreas, hydrolyze peptides one residue at a time from the C-terminal end. Carboxypeptidase A preferentially cleaves peptide bonds when an aromatic amino acid is the C-terminal residue. Carboxypeptidase B prefers basic residues. Because these enzymes sequentially cleave peptide bonds starting at the C-terminal residue, the first amino acid liberated is the C-terminal residue. 

Saccharic acids. See Aldaric acids Saccharin, 997 Salicylic acid, 737 acetylation of, 952 acidity of, 953 synthesis of, 952-954 Samuelsson, Bengt, 1025 Sandmeyer reactions, 892, 894, 906—907, 919 Sanger, Frederick, 1070—1074, 1101—1102 Sanger s reagent. See l-Fluoro-2,4-dinitrobenzene a-Santonin, 1046 Saponification, 794—799 Sawhorse diagrams, 90—91 Saytzeff. See Zaitsev, Alexander M. Schiemann reaction, 892, 893, 905 Schiff s base, 673, 689. See also Imines Schrbdinger, Erwin, 7 Schrbdinger equation. See Wave equation Scientific method, 217... 

Aryl fluorides with two nitro groups are very reactive toward nucleophilic aromatic substitution. The reaction of l-fluoro-2,4-dinitrobenzene, known as Sanger s reagent, with the amino acid phenylalanine occurs at room temperature. This reaction forms the basis of a method used in the analysis of proteins that is described in Section 25.11. 

Use of DNP-amino acids, formed by condensation of l-fluoro-2,4-dinitrobenzene (FDNB) with the free amino group of an amino acid, was first described by Sanger in 1945 (83). Sanger identified DNP-amino acids by paper chromatography. Since then many modifications in the methods of obtaining derivatives of amino acids for sequence analysis and in identification of such derivatives have been reported, and the use of DNP-amino acids for sequencing purposes is rapidly going out of date. Nevertheless, the importance of DNP-amino acids is not yet lost. In view of the limited applications of DNP-amino acids at present, the methods of preparation of these derivatives from standard amino acids or peptides are not described here. However, the details of those procedures can be obtained from Rosmus and Deyl (88) and Bailey (146). 

Studies of the structure of papain were initiated by Thompson (165), who determined the amino or N-terminal amino acid sequence and the free amino groups. Crystalline papain and mercuripapain were allowed to react with l-fluoro-2,4-dinitrobenzene (FDNB) by the procedures of Sanger (136). The dinitrophenyl (DNP) proteins were subsequently hydrolyzed in 6 iV HCl and the DNP-amino acids isolated by partition chromatography of the ether extracts of the hydrolyzates. Quantitative estimates of the DNP-amino acids were made spectrophotometrically. The results are given in Table V. 

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