Phenyl isothiocyanate, Edman reagent

The final step in the degradation of uracil requires the presence of /3-ureidopropionase, the enzyme that catalyzes the hydrolysis of TV- carbamoyl-/3-alanine to /3-alanine, CO2, and ammonia. This assay measures the phenyl-isothiocyanate (Edman reagent) derivative of /3-alanine. 

Edman s reagent phenyl isothiocyanate. A reagent used to determine the N-terminal amino acid of a polypeptide. 

A more useful procedure, in that it allows sequential determination of the A-terminal amino acids in a peptide, is the Edman degradation. This process removes the A-terminal amino acid, but leaves the rest of the chain intact, so allowing further reactions to be applied. The reagent used here is phenyl isothiocyanate. 

Phenyl isothiocyanate is also used for peptide sequencing in combination with colored Edman s reagent 4-W-,/V -dimethy]aminoazobenzene-4 -isothiocyanate (DABITC). Prieto (48) used this DABITC-PITC procedure to determine the partial terminal N-NH2 sequence of the low-molecular-weight peptide fraction from bread dough and bread. 

By the primary structure of a peptide or protein, we mean its amino acid sequence. Complete hydrolysis gives the amino acid content. The N-terminal amino acid can be identified by the Sanger method, using 2,4-dinitrofluorobenzene. The Edman degradation uses phenyl isothiocyanate to clip off one amino acid at a time from the N-terminus. Other reagents selectively cleave peptide chains at certain amino acid links. A... 

In the sequential degradation according to the Edman procedure methyl or phenyl-isothiocyanate is used as the reagent and the reaction starts from the amine end of the... 

An even simpler protocol is employed in the formation of A-phenylthiocar-bamoyl derivatives (PTC-amino acids, C6H5NHCSNHCHRC02H Figure 4.1) by reaction in a suitable buffer with the Edman reagent, phenyl isothiocyanate (West and Crabb, 1989). 

Edman s method of stepwise degradation (Edman, 1949, 1950) (Scheme 5.3) involves reaction of the a-amino group at the TV-terminus of a peptide with phenyl isothiocyanate under slightly basic conditions. Excess reagent is extracted and the... 

There are several ways to identify the N-terminal amino acid of a peptide or protein. One of the most widely used methods is to treat the protein with phenyl isothiocyanate (PITC), more commonly known as Edman s reagent. This reagent reacts with the N-terminal amino group, and the resulting thiazolinone derivative is cleaved from the protein under mildly acidic conditions. The thiazolinone derivative is extracted into an organic solvent and in the presence of acid, rearranges to a more stable phenylthiohydantoin (PTH). 

In the sequencing of a peptide, the Edman reagent, phenyl isothiocyanate, reacts with the peptide s N-terminal residue. The modified amino acid can be cleaved off, leaving the rest of the peptide intact, and can be detected as the phen-ylthiohydantoin derivative of the amino acid. The second amino acid of the original peptide can then be treated in the same way, as can the third. With an automated instrument called a sequencer (Figure 5.20), the process is repeated until the whole peptide is sequenced. 

In Edman degradation, a polypeptide is shortened one amino acid at a time using the reagent phenyl isothiocyanate. (18.5)... 

The remaining peptide has another amino unit, and it is treated with more phenyl isothiocyanate and then cleaved to examine and identify the next amino acid. If a pentapeptide (ala-val-ser-leu-ile) is subjected to the sequential Edman degradations by treatment with Edman s reagent and loss of the phenylthiohydantoin, each amino acid can be identified, in order. Because the process begins at the N-terminus and progresses toward the C-terminus, both the identity of the amino acids and their exact sequence in the peptide are known. This method can be used to identify from 30 to 60 amino acid residues in a long peptide under the right conditions. 

The Edman degradation involves removing one amino acid residue at a time and identifying each residue as it is removed. The process is repeated until the entire peptide has been sequenced. To perform an Edman degradation, a peptide is first treated with phenyl isothiocyanate followed by trifluoroacetic acid. These two reagents remove the amino acid residue at the N terminus and convert it into a phenylthiohydantoin (PTH) derivative. 

Several different methods can reveal the identity of the residue at the amino end. Most exploit the nniqueness of the free amino substiment, which may enter into specific chemical reactions that serve to tag the A -terminal amino acid. One such procedure is the Edman degradation, and the reagent used is phenyl isothiocyanate, CgH5N=C=S (a snlfur analog of an isocyanate Section 20-7). 

These methods are, however, completely destructive of the rest of the peptide chain—a single piece of information is gained at the cost of significant material use. When the Af-terminus of a peptide is reacted with phenyl isothiocyanate, PhN=C=S, the Edman reagent, a phenylthiohy-dantoin, 22.39, is obtained and the rest of the peptide chain is left intact (Figure 22.36). So the process can be repeated, and this methodology is the basis for automatic sequenators. The phen-ylthiohydantoin contains the R group from the Af-terminal amino acid and can thus be identified. 

A-terminal labelling with phenyl isocyanate, which is a fast and quantitative reaction also applied during Edman degradation [100]. An alternative reagent is 4-sulfophenyl isothiocyanate [101]. 

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