PITC determination

Phenyl isothiocyanate is also used for peptide sequencing in combination with colored Edman s reagent 4-W-,/V -dimethy]aminoazobenzene-4 -isothiocyanate (DABITC). Prieto (48) used this DABITC-PITC procedure to determine the partial terminal N-NH2 sequence of the low-molecular-weight peptide fraction from bread dough and bread. 

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. 

Determining the structure of a peptide or protein is carried out in several steps. The identity and amount of each amino acid present in a peptide is determined by amino acid analysis. The peptide is hydrolyzed to its constituent a-amino acids, which are then separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forma an easily identifiable phenylthvobydantoin (PTH) derivative of the N-terminal amino acid. A series of sequential Edman degradations allows the sequencing of a peptide chain up to 50 residues in length. 

Several techniques have been developed to analyse amino acids. The following procedure describes one of the most frequently used methods for the determination of amino acid concentration, the PITC method. Free amino acids in medium samples are derivatized with phenylisothiocyanate (PITC) to produce phenyl-thiocarbamyl (PTC) amino acids. After separation by reverse-phase HPLC, the PTC amino acids are detected using a UV spectrophotometer at 254 nm. 

Before running the acquisition on a flow cytometer, we usually calibrate the machine using the three-color BD CaliBRITE kit containing unlabeled beads, fluorescein isothiocyanate (PITC)-labeled beads, phycoerythrin (PE) labeled beads, and peridin chlorophyll protein (PerCP)-labeled beads. In addition, we add the allophycocyanin (APC)-labeled beads to setup all four colors used in the assay. This procedure allows determining the correct settings (compensation and detector/amplitude) when several colors are simultaneously used. 

Figure 3.3 shows a small portion of a mass spectrum of PFK and an organic compound. The most intense peak near mass 169 in Figure 3.3a is from the PITC. The other smaller peaks are from an unknown chemical. The peaks are centroided, and a new calibration table is generated from PFK ions in the spectrum. The accurate masses can then be determined, as shown in Figure 3.3b. The peak labeled with 169.0897 is shown to be CijHjjN, which has an exact calculated mass of 169.0891. The smaller peak has a composition of CnHyON, which has an exact calculated mass of 169.0528.

Isothiocyanates are widely used for derivatizing primary amines. They possess higher selectivity and are less reactive towards water, hydroxyl and carboxylic groups compared with isocyanates [42-44]. Labelling with phenyl isothiocyanate (PITC) or naphthyl isothiocyanate (NITC) has been used for the determination of C3—Cjo alkylamines in ethanol [45]. 

Both phenyl isocyanate (PIC) and phenyl isothiocyanate (PITC) are used as derivatization reagents for aliphatic amines that lack strong UV-chromaphores to improve detectability and retention on a reversed-phase HPLC column. PIC is for both primary and secondary amines, while PITC is mainly for primary amines (14). They are used to determine extraction efficiencies of individual amines. 

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