Amino acid phenylthiocarbamylated

Other abbreviations used in this review ACE, angiotensin converting enzyme ANP, atrial natriuretic peptide DADL, (D-Ala -D-Leu)-enkephalin DAP, dipeptidylaminopeptidase GABA, gamma-aminobutyric acid PTC-amino acid, phenylthiocarbamyl-amino acid. 

Vasanits, A. and Molnar-Perl, I., Temperature, eluent flow-rate and column effects on the retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography, J. Chromatogr., A, 832,109, 1999. 

Zahou, E., Jornvall H., and Bergman T., Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation, Anal. Biochem., 281, 115, 2000. 

I. Molnar Perl, Advances in the High Performance Liquid Chromatographic Determination of Phenylthiocarbamyl Amino Acids, Journal of Chromatography, A, 661, 43 50 (1994). 

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. 

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. 

KL Woo, QC Hwang, HS Kim. Determination of amino acids in the foods by reversed-phase high-performance liquid chromatography with a new precolumn derivative, butylthiocarbamyl amino acid, compared to the conventional phenylthiocarbamyl derivatives and ion-exchange chromatography. J Chromatogr A 740 31-40, 1996. 

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. 

Another widely used method for qualitative and quantitative analysis of amino acid mixtures is high-performance liquid chromatography (HPLC) (see Experiments 2 and 6). The mixture of amino acids is first subjected to reaction with phenylisothiocyanate (PITC) to convert them to the phenylthiocarbamyl-amino acid derivatives, which are then subjected to chromatographic separation. The derivativatization of the amino acids serves two purposes it attaches a UV-absorbing tag, which makes their quantitative determination easy, and it converts them to a more hydrophobic form, which is necessary for good separation on the reverse-phase system commonly used with this technique. This method of amino acid analysis will be used in Experiment 6. 

Phenylthiocarbamyl (PTC) derivatives were obtained at pH 12 by in situ reaction for 15 min or in a tube of phenylisothiocyanate with the amino group of amino acids and separated on silica gel 60 F254 and RP-18 F254 plates with 2D and ID chromatography, respectively. 

The three general approaches to enantiomer separation entail a chiral stationary phase, a chiral mobile phase, or a chiral reagent. Tandem columns, with reversed and chiral stationary phases, were used to separate 18 d-l pairs of phenylthiocarbamyl (PTC)-amino acids in 150 min. OPA-amino acid enantiomers have been separated on both ion-exchange and reversed-phase columns using a sodium acetate buffer with an L-proUne-cupric acetate additive. Chiral reagents, such as Marphey s reagent and OP A/ IBLC (iV-isobutyryl-L-cysteine), were successfully used for racemization analysis within 80 min. 

Ammo acid analysis of purified rCRALBP is useful to quantify the protein, and for corroborating the identity of the recombinant protein (Table 1). We typically perform phenylthiocarbamyl (PTC) amino acid analysis on about 1 pg rCRALBP samples (20-30 pmol protein per analysis) using Applied Biosystems instrumentation (26). 

Palego L, Giannaccini G, Saccomanni G, Ross A, Lucchesi V, Mascia G, et al. Modified RP-LC of phenylthiocarbamyl amino acid adducts in plasma acetonitrile extracts using multiple internal standards and phot-diode UV detection. Chromatographia 2010 71 291-7. 

Step 2. Synthesis of amino acid derivatives. The amino acids released by hydrolysis of the protein are converted to phenylthiocarbamyl (PTC) amino acids by reaction with phenyhsothiocyanate (PICT). 

By the DNP and PTC (phenylthiocarbamyl) methods, the N-terminal amino acid was determined to be proline and the N-terminal amino acid sequence to be H-Pro Arg Arg . The alternate action of carboxypeptidases B and A on clupeine YII followed by hydrazinolysis indicated that the amino-acid sequence at the C-terminal region was Pro Arg2 (Val, Ser) Arg4 Ala Arg4-OH. 

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