Dinitrophenyl proteins, hydrolysis

FDNB reacts with all free -NH2 groups, including e-amino groups. Following acid hydrolysis of the dinitrophenylated protein, a separation of the dinitrophenylated N-terminal amino acid from the rest of the hydrolysis products can be achieved by organic solvent extraction of the hydrolysate. A more modern procedure utilizes dansyl chloride instead of FDNB. This method requires less protein, because dansylated amino acids can be identified via fluorescence measurements. 

Peptide analysis. The reagent was introduced by Sanger for identification of the amino-terminal group of a protein or peptide. Condensation occurs under mild conditions to form a 2,4-dinitrophenyl protein on acid hydrolysis the terminal... 

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. 

Alternatively, MW-assisted acid hydrolysis using 6 m HC1 at 100-110°C under anaerobic conditions can be used to hydrolyze proteins [32]. Conflicting results as to the stability of selenomethionine have been reported in the literature [133, 140-142]. Stability is probably dependent on sample matrix and procedural variations. Selenocysteine is widely reported to be unstable [143], and normally is reacted with either l-fluoro-2,4-dinitrobenzene, iodoacetic acid, or ethylenimine to form a stable 2,4-dinitrophenyl, carbomethyl or aminoethyl derivative before hydrolysis [137, 144-146]. 

An important example ol this behaviour is provided by the reaction of 1-f1uoro-2,H-dinitrobeni eno ai with the terminal amino group of proteins Subsequent acidic hydrolysis yields the yeliow 2.4-dinitrophenyl derivative of the terminal amino acid ol the protein, which can then be identified. With the help of this technique of end-group analysis, Sanger was able to determine the primary structure o insulin. 

Determination of the completeness of lysine acylation is performed by dinitrophenylation (Fraenkel-Conrat et al. 1955) of the succinylated protein, followed by acid hydrolysis, and amino acid analysis. Since fi-dinitrophenyllysine is stable to acid hydrolysis, the recovery of lysine reflects the -succinyllysine content of the protein. 

The determination of AT-terminal amino acids of polypeptides or proteins by means of reaction with 2,4-dinitrofluorobenzene is used as a chemical method for the estimation of molecular weight. This operation is rather complex because of the need for hydrolysis, separation of DNP-amino acids, and colorimetric comparison with a standard curve. Schiedt and Restle (1954) have estimated the degree of polymerization of oligopeptides using dinitrophenylation and infrared spectroscopic... 

As shown in Table 1.5, betaines are widespread in both the animal and plant kingdoms. Derivatization of amino acids by reaction with l-fluoro-2,4-dinitrobenzene (FDNB) yields N-2,4-dinitrophenyl amino acids (DNP-amino acids), which are yellow compounds and crystallize readily. The reaction is important for labeling N-terminal amino acid residues and free 8-amino groups present in peptides and proteins the DNP-amino acids are stable under conditions of acidic hydrolysis (cf. Reaction 1.33). 

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