Amino acid phenylisothiocyanate

Gas chromatography (GC) for amino acid analysis is the alternative to HPLC that has found the greatest acceptance. It requires the preseparation derivatization of the amino acids to render them volatile. For this purpose, amino acids are frequently converted into acylated esters. N-Trifluoroacetyl-n-butyl esters and /V-heptafluorobutyrylisobutyl esters are most commonly employed. There have been comparative studies (3,4) that document similar (if not equivalent) analytical results for GC and the classic ion-exchange chromatographic method applied to a variety of food samples. Comparison (5) of GC to the reversed-phase HPLC determination of amino acids (phenylisothiocyanate derivatized) also shows excellent agreement. 

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. 

The mechanism involves the initial formation of a substituted urea followed by ring closure to form the thiohydantoin. The amino acid is dissolved in 60% aqueous pyridine containing the phenylisothiocyanate... 

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. 

Heinrikson, R.L. and Meredith, S.C. 1984 Amino acid analysis by reverse-phase high-performance liquid chromotography Precolumn derivatization with phenylisothiocyanate. Analytical Biochemistry 136 65-74. 

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 mol l-1 HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acid identified by comparison to phenylthiohydantoin derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established...

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

Naulet et al. [223] compared three chromatographic techniques lEC on an automatic amino acid analyzer, HPLC (with phenylisothiocyanate derivatization) and GC for the determination of the free amino acid content in 64 orange juices from different countries. The consistency of the different methods was estimated by considering the mean standard deviation for the set of amino acids observed. The best consistency was observed between lEC and HPLC. 

To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 3-25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluo-roacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The deriva-tized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over... 

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... 

In modem sequencing methods vinylpyridine, which reacts in a similar way, is often used. It can be detected during amino acid analysis or sequencing after derivatization with phenylisothiocyanate (Eq. 3-30). 

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. 

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. 

PITC (phenylisothiocyanate) Aabs = 254 nm. Phenylthiocarbamyl amino acid derivatives are moderately stable at room temperature (1 day). PITC reacts well with both primary and secondary amino acids. Reaction time is approximately 5 minutes at room temperature. Excess reagent must subsequently be removed under vacuum. Also, for hydrolyzed samples, hydrochloric acid must be completely removed prior to derivatization. As a result, even though the actual reaction time is reasonably fast, the total time for various sample manipulations can add up to 2 hours. This is partially compensated by the extremely fast separation possible (12 minutes). Detection is by UV absorption only. Detection limits are typically in the high picomole range. Short column life can result due to unreacted PITC getting into the column. Unlike some of the other reagents, PITC quantifies tyrosine and histidine very well. PITC analysis is available as a commercially prepackaged system dubbed Pico-Tag by Waters Corporation. Representative references include 184-188. See Fig. 11 for a typical separation. 

SA Cohen, DJ Strydom. Review amino acid analysis utilizing phenylisothiocyanate derivatives. Anal Biochem 174 1-16, 1988. 

JF Davey, RS Ersser. Amino acid analysis of physiological fluids by high-performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography. J Chromatogr Biomed Appl 528 9-23, 1990. 

DJ Strydom, SA Cohen. Comparison of amino-acid analyses by phenylisothiocyanate and 6-amino-quinoly 1-jV-hydroxysuccinimidyl carbamate precolumn derivatization. Anal Biochem 222 19-28, 1994. 

RW Beaver, DM Wilson, HM Jones, KD Haydon. Amino acid analysis in feed and feedstuffs using precolumn phenylisothiocyanate derivatization and liquid chromatography—preliminary study. J Assoc Off Anal Chem 70 425-428, 1987,... 

RG Elkin, AM Wasynczuk. Amino acid analysis of feedstuff hydrolysates by precolumn derivatization with phenylisothiocyanate and reversed-phase high-performance liquid chromatography. Cereal Chem 64 226-229, 1987. 

SR Hagen, B Frost, J Augustin. Precolumn phenylisothiocyanate derivatization and liquid chromatography of amino acids in food. J Assoc Off Anal Chem 72 912-916, 1989. 

G Sarwar, HB Botting, RW Peace. Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives. J Assoc Off Anal Chem 71 1172-1175, 1988. 

SF Shang, H Wang. Sensitive determination of amino acids in kelp by reversed phase high performance liquid chromatography with precolumn derivatization using phenylisothiocyanate. Chro-matographia 43 309-312, 1996. 

MJ Gonzalez-Castro, J Lopez-Hernandez, J Simal-Lozano, MJ Oruna-Concha. Determination of amino acids in green beans by derivatization with phenylisothiocyanate and high-performance liquid chromatography with ultraviolet detection. J Chromatogr Sci 35 181-185, 1997. 

M Calull, J Fabregas, RM Marce, F Borrull. Determination of free amino acids by precolumn derivatization with phenylisothiocyanate. Application to wine samples. Chromatographia 31 272-276, 1991. 

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. 

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