3,5-Dinitrobenzene, detection reagent

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. 

For the detection of ketosteroids, an m-dinitrobenzene solution can be used (17-keto-steroids, violet 3-keto-A" groups, blue). A" -3-Oxo steroids and A" -3-oxo steroids can be distinguished using a phthalic acid-/7-phenylene-diamine reagent, which gives yellow and orange-brown colors, respectively. Detailed discussion of the... 

The steroid moiety can be detected once again with sulphuric acid or, for 17-ketosteroid conjugates, with m-dinitrobenzene for example. The acid component can be characterised by either the Folin-Ciocalteu reagent (No. 122) or pyridylazonaphthol reagent (No. 215) for glucuronides and methylene blue (Rgt. No. 162) for sulphates [44]. 

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