Phenylisothiocyanate protein sequencing

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

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... 

Amino acid sequencing may be carried out in a number of ways. The most widely used is the Edman degradation procedure in which phenylisothiocyanate is used to react with the amino acid residue at the amine end of the protein chain. This derivatized residue is removed from the remainder of the protein and converted to a phenylhydantoin derivative which is identified by using, for example, HPLC. 

Phenylisothiocyanate is used to sequence proteins from the N terminus, because no destruction of the protein during the determination is necessary. 

If the phenylisothiocyanate method is used, the cyclization and release of the N-terminal derivative occurs under mild conditions that leave the rest of the chain intact. It is therefore possible to take the protein chain, now without its original N-terminal residue, and repeat the procedure to determine the second residue in the sequence, and so on. Unfortunately, at each step, there is a finite chance of additional peptide hydrolysis or incomplete reaction, and uncertainty tends to accumulate after 10 to 20 cycles. 

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... 

Derivatization of amino acids with phenylisothiocyanate (XXXXII) to give phenylthiohydantoins (XXXXIIl) is utilized in the Edman method for sequence determination of peptides and proteins. The first step, performed under mildly basic conditions, may be regarded in principle as an acylation ... 

J. Y. Chang, D. Brauer and B. Wittman-Liebold, Micro-Sequence Analysis of Peptids and Proteins using 4-NN-dimethyl-aminoazobenzene 4 -isothiocyanate/phenylisothiocyanate Double Coupling Method, FEBS Letters, 93 205-214 (1983). 

It might be noticed that under acidic conditions (i.e., anhydrous trifluoro-acetic acid) the hydroxyl (protonated) becomes a leaving group, and so it is expected an amine (amide linkage) could also leave. Thus, the reaction of a protein with phenylisothiocyanate provides an important method for the determination of the N-terminal amino acid and subsequent determination of the amino acid sequence of the protein. Remembering that the monomeric units of the protein are connected by amide (so-called peptide) linkages, this may be illustrated for the simple dipeptide glycylalanine. 

Figure 18.5. MALDI-TOF mass spectrum from protein ladder sequencing of the N-terminal peptide of cofilin (G-S-R-S-G-V-A-...). A stepwise degradation was carried out in the presence of 5% phenylisocyanate/95% phenylisothiocyanate. The peptide was subjected to two cycles of ladder generating chemistry. ...

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