Dansyl-Edman method

Today s sequence techniques have achieved 9-10 orders of magnitude higher detection limit for PTH-amino acid than Edman s first report, in which he described the sequence of Ala-Leu-Gly with 100 pmol. Matsuo et al. succeeded in determing the structure of FSH-RH (LH-RH), a decapeptide hormone, in 1971 by using the Dansyl Edman method.83 This historical work used 200 nmol of FSH-RH purified from 165,000 porcine hypothalami and led a principal investigator, A. Schally, to win the Nobel Prize. Today, we would only need less than one hundred porcine hypothalami to complete the same job on a state-of-art sequencer. Those who plan the structural analysis of even barely detectable amounts of a peptide or protein are urged to start the plan such study could open up a new area in biochemistry. 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

B 1. Study the details of the Edman and dansyl chloride methods for amino-terminal analysis of proteins. Discuss the advantages and disadvantages of each method. 

When the major labeled thermolytic peptide was sequenced by the dansyl-Edman and Edman methods, it was shown to have the sequence Val-X-Tyr, and 99% of the label was released at the second position (X), corresponding to a derivative of Glu-148. Analysis of a labeled chymotryptic peptide (residues 141-149) gave the expected sequence Ala-Glu-Gly-Ser-Ser-Ser-Val-X-Tyr, and most of the associated radioactivity... 

The chemical structures of these peptides were established in the usual way by quantitative amino-acid analysis and sequence analysis by a combination of chemical and enzymic methods, including dinitrophenylation, dansylation, Edman degradation, hydrazinolysis and digestion with leucine aminopeptidase and carboxypeptidases A and B. The Neutral Protease peptides of the salmine and iridine components or derivatives thus identified are listed in Table VIII-6 with their recovery values. Differences in the amino-acid sequences and amounts of the peptides obtained from iridine I aroused the suspicion that two molecular species (a and b) were present in the iridine I component. This result, as already described, was supported by observations on the thermolysin peptides of iridine I. 

Edman has also devised the highly sensitive "dansyl" method in which a peptide is treated with 5-dimethyl-aminonaphthalenesulfonyl chloride, followed by acidic hydrolysis. A derivative of the N-terminal residue is obtained which can be followed during its analysis by virtue of its characteristic fluorescence. What is the derivative Why does it survive the acid treatment that cleaves the peptide bonds ... 

End group analysis, determination of N-terminal (— dinitrophenyl method, —> dansyl method, Edman degradation) and C-terminal C-terminal end group analysis, Akabori method) amino acid residues in the primary structure analysis of peptides and proteins. 

Derivatization of free amino group of amino acids with 5-methyl amino naphthalene-1-sulfonyl (dansyl) chloride has become increasingly popular for N-teiminal end determinations in proteins and for manual Edman degradation (91). In addition, dansylation reaction has also been used as one of the most sensitive methods for quantitative amino acid analysis (117,118). 

Improved methods for end group analysis have become available, notably the use of dansyl chloride (5-dimethylaminonaphthalene-l-sulphonyl chloride) for labelling iV-terminal amino acids and the Edman technique. Dansyl-amino acids fluoresce strongly in ultraviolet light and can be detected in very small amounts so that the method is much more sensitive than Sanger s original DNP technique. 

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