Phenylisothiocyanate Edman reagent

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)... 

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. ... 

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... 

The guanidinated inhibitor retained only one free amino group, which was on the terminal leucine. The absence of other reactive groups made it possible to subject this derivative to the Edman degradation. This in turn made it possible to determine the role of the amino acids near the amino terminus in the inhibiting activity. Four steps of Edman degradation were carried out as described in EXPERIMENTAL PROCEDURE. Another portion of the material was used as a control of nonspecific loss of activity due to exposure to the Edman reagents this sample was subjected to identical treatment except for omission of the phenylisothiocyanate. 

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. 

Sequencing is a stepwise process of identifying the specific amino acids at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman s reagent, is... 

The Edman degradation method for polypeptide sequence determination. The sequence is determined one amino acid at a time, starting from the amino-terminal end of the polypeptide. First the polypeptide is reacted with phenylisothiocyanate to form a polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino-terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected spectrophoto-metrically. The remaining intact polypeptide, shortened by one amino acid, is then ready for further cycles of this procedure. A more sensitive reagent, dimethylaminoazobenzene isothiocyanate, can be used in place of phenylisothiocyanate. The chemistry is the same. 

The primary structure (i.e., the amino acid sequence) of a protein can be determined by stepwise chemical degradation of the purified protein. By far the most powerful and commonly used technique for doing this is the automated Edman degradation. The amino terminal amino acid residue of the polypeptide is reacted with Edman s reagent (phenylisothiocyanate) to form the phenylthiocar-bamyl derivative, which is removed without hydrolysis of the other peptide bonds by cyclization in anhydrous acid. The amino acid derivative is converted to the more stable phenylthiohydantoin and identified by HPLC. The process can be repeated many times, removing the amino acids from the amino terminus of the polypeptide one residue at a time and identifying them until the entire sequence... 

Each fragment is sequenced through repeated cycles of a procedure called the Edman degradation. In this method phenylisothiocyanate (PITC), often referred to as Edman s reagent, reacts with the N-terminal residue of each fragment. 

Decisive progress in stepwise degradation was brought about by Pehr Edman (Plate 15) in 1950 [11], who found that phenylisothiocyanate, the sulfur analog of Bergmann s reagent, reacts with the N-terminal amino group at pH 9 even at room temperature, and that the thioureido compound hereby formed is readily split by weak acids, e.g. trifluoroacetic acid, to yield a 2-anilinothiazolin-5-one. 

The new N-terminal residue can then be removed as a phenylhydantoin in a second cycle of phenylcarbamoylation and cyclization. This method of stepwise degradation, while discovered several decades earlier (Bergmann et al. 1927), reached practicality only in 1950 when Edman modified the reagent and applied chromatographic procedures for identification of the cyclic products. The improved reagent, phenylisothiocyanate, smoothly converts the peptide to the phenylthiocarbamoyl derivative which is cyclized and cleaved by the action of hydrochloric acid (dissolved in an organic solvent such as dioxane) ... 

Fluorescein isothiocyanate was proposed by Maeda el al. as a fluorescent end-group reagent [218] analogous to phenylisothiocyanate in the Edman method. A procedure for the determination of free amino acids by formation of fluorescent thiohydantoins with fluorescein isothio-cytinate was reported by Kawauchi ef al. [219]. 

Dimethylamino-l-naphthylisothiocyanate [224], 4-(benzyloxycarbonylaminomethyl)phenylisothiocyanate [225] and 4-(dimethylaminonaphthalene-l-sulphonyl-amino)phenylisothiocyanate [226, 227] have been introduced in order to improve the sensitivity of the Edman method. These reagents are used in the same way as the other isothiocyanates, and allow end-group determinations in the picomole-range. 

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