Insulin dinitrophenylation

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. 

Insulin was reacted with Sanger s reagent and then completely hydrolyzed. Two amino acids, a glycine and a phenylalanine, were found to be labeled with the 2,4-dinitrophenyl group. 

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. 

In the case of the respiratory proteins, combination with oxygen has been used to determine minimal molecular weights. A method used for the hormone insulin was to cause it to combine with the reagent l-fluoro-2,4"dmitrobenzene. There was a complication in that both monosubstituted and disubstituted products were obtained, but the monosubstituted material could be separated and analyzed for the dinitrophenyl group. The result was that 6000 g of protein contained 1 mol of the dinitrophenyl group the minimum molecular weight of insulin is therefore 6000 g mol h... 

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