PTH derivative

Step 3 Once formed the thiazolone derivative isomerizes to a more stable phenylthiohydantom (PTH) derivative which IS isolated and characterized thereby providing identification of the N terminal ammo acid The remainder of the peptide (formed m step 2) can be isolated and subjected to a second Edman degradation... 

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

Edman degradation (Section 27 13) Method for determining the N terminal amino acid of a peptide or protein It in volves treating the material with phenyl isothiocyanate (CgH5N=C=S) cleaving with acid and then identifying the phenylthiohydantoin (PTH derivative) produced Elastomer (Section 10 11) A synthetic polymer that possesses elasticity... 

FIGURE 5.19 N-Tertninal analysis using Edman s reagent, phenylisothiocyanate. Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinone derivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treatment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative. 

What is the N-terminal residue on a peptide that gives the following PTH derivative on Edmau degradation ... 

Draw the structure of the PTH derivative that would be formed on Edman degradation of angiotensin II (Problem 26.12). 

Refer to Figure 26.2 and propose a mechanism for the final step in the Edrnan degradation—the acid-catalyzed rearrangement of the ATZ derivative to the PTH derivative. 

The phenylthiohydantion (PTH) derivative plus the AA backbone has a mass of 191 Daltons. Thus, subtracting 191 from the molecular... 

Multiple development techniques using stepwise solvent gradients enable a subset of optimal separation conditions to be used to separate a mixture of wide polarity that cannot be separated using a single mobile phase (117,119,120,125). As an example of this approach the separation of 20 common protein amino acid PTH-derivatives is shorn in Figure 7.12 (126). Five... 

Finally, in a third stage — conversion — the unstable thiazolinone is converted into a phenylthiohydantoine (PTH) derivative which can be identified. 

The unstable thiazolinones are converted into stable hydantoines in order to facilitate their identification. Conversion and identification are carried out outside the instrument after extraction of the thiazolinones with butylchlor-ide. The conversion reaction as well as the problems associated with identification of the PTH-amino acids were studied in detail by Edman and described explicitly in Needleman s book on Protein Sequence Determination Conversion is generally carried out in 1 N HCl at 80 °C within 10 min. The PTH-derivatives are extracted from the aqueous phase with ethyl acetate with the exception of PTH-arginine, PTH-histidine and PTH-cysteine which remain in the aqueous phase. 

In view of its rapidity we found thin layer chromatography convenient for identification of the amino acids liberated by the first 20—30 degradation cycles. For identification of PTH-derivatives from additional degradation steps we prefer gas-liquid chromatography because of its merits mentioned above, particularly its greater sensitivity. Several colorimetric reactions and chromatographic systems are available for the identification of those PTH-amino acids which remain in the aqueous phase when the PTH-derivatives are extracted with ethyl acetate 23.24,25,13) our hands, thin layer electrophoresis was found to be satisfactory 26,27)... 

Eyem and Sjoquist have suggested the use of short (4.5 m) glass capillary columns and reported the separation of 19 of 20 silylated methyl thiohydantoin (MTH) derivatives of the amino acids (62). The histidine derivative can be separated on the same column by starting at a higher temperature. Separation of some of the silylated PTH derivatives was also demonstrated, though not in as much detail as the MTH derivatives. 

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. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

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