Edman reagent

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

An alternative approach for the analysis of blood samples from the same group of Iranian mustard gas victims has been described (50). As mustard gas alkylates amino acids in hemoglobin, adducts will be formed, which remain in the bloodstream for some time. Selective cleavage of the alkylated N-terminal valine of the a-chain of hemoglobin was carried out by using the modified Edman reagent pentafluorophenyl isothiocyanate. After derivatiza-tion of the adduct-derived pentafluorophenylthio-hydantoin with heptafluorobutyric anhydride, the... 

If you had a peptide with the following probable structure, how would you proceed to prove it Be sure to indicate all separation procedures that are likely to work in any specific instance, as well as cleavage reagents. Do not use the Edman reagent or the carboxypeptidases beyond the C terminus of any peptide. 

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. 

Contained ala + gly + cys + glu + arg + ile + N HT Carboxypeptidase A liberated isoleucine Treatment with phenylisothiocyanate (PITC, the Edman reagent) yielded the phenylthiohydantoin derivative of glycine (PTH-glycine)... 

Deduce the sequence of amino acids in a peptide from the following information (a) composition = phe4- pro+gIu-l-2 lys (b) treatment with the Edman reagent yielded PTH-glutamale and (c) trypsin, car-boxypeptidase A, and carboxypeptidase B did not release any smaller peptides or amino acids. 

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). 

The precolumn technique that is most frequently employed today was developed during the early 1980s [32,33]. For this method, The classical Edman reagent phenylisothiocyanate (PITC) is used for amino acid derivatization after hydrolysis. Separation of the PTC amino acids i then accomplished by HPLG, with detection at 254 nm. Although standard Cig columns available Irom a variety of vendors are suitable for separation of the PTC-derivatized amino acids, there are specific columns that have bqen optimized for this purpose (e.g.. Waters). Approximately 0.5 /ug of peptMe should be hydrolyzed for analyses using precolumn derivatization. ... 

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. 

Reflect and t ply Why can the Edman degradation not be used effectively with very long peptides Hint Think about the stoichiometry of the peptides and the Edman reagent and the percent yield of the organic reactions involving them. 

Reflect and Apply What would happen during an amino acid sequencing experiment using the Edman degradation if you accidentally added twice as much Edman reagent (on a per-mole basis) as the peptide you were sequencing ... 

The amount of Edman reagent must exactly match the amount of N-termini in the first reaction. If there is too little Edman reagent, some of the N-termini will not react. If there is too much, some of the second amino acid will react. In either case, there will be a small amount of contaminating phenylthiohydantoin (PTH) derivatives. This error grows with the number of... 

Phenylisothiocyanate (PITC). The use of PITC (Edman Reagent) to form thiohydantoin (PTH) derivatives of amino acids for protein sequencing is well known. The development of reversed phase systems for the separation of these PTH derivatives has resulted in the introduction of both isocratic and gradient elution systems (Fig. 11.2.11). A microbore HPLC unit for PTH-amino acid analysis is now available along with the recently introduced gas phase sequenator... 

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