PTC amino acid

West, K.A. and Crabb, J.W 1990 Performance evaluation automatic hydrolysis and PTC amino acid analysis. In Vallafraca, J.J., ed.. Current Research in Protein Chemistry Techniques, Structure, and Function. San Diego, Academic Press 37-48. 

Hardware requirements for the separation of PTC-amino acids are similar to those for OPA amino acid determination. Since the derivatives do not fluoresce the technique is limited to UV detection, which normally takes place at 245nm. 

PITC Amino acid PTC-amino acid Simplified reaction between PITC and amino acids... 

Day 3 Analysis of the Paper Chromatogram and Preparation and Analysis of PTC-Amino Acids... 

Dry the PTC-amino acids in the Speed-Vac under the low heat setting. 

Inject 100 pX of the PTC-amino acid sample and apply the following mobile-phase gradient during development (mark the time of injection, or T = 0, on the chart recorder) ... 

Detect the PTC-amino acids as they elute with a spectrophotometer set at 254 nm. When the signal output (10 mV maximum) is relayed to a chart recorder with a speed of 0.5 cm/min, the PTC-amino acid peaks will show good height and good resolution. 

Inject a 100-jul sample of the 20 PTC-amino acid standards and apply the same mobile-phase gradient. The sequence of the PTC-amino acids eluting from the column developed with this gradient is shown in Table 6-1. 

Measure the retention times (Rt) of the unknown PTC-amino acids and compare these to those of the PTC-amino acid standards. The retention time (Rt) can be calculated as follows ... 

Table 6-1 Retention Times of PTC-Amino Acids on a C18 Reverse-Phase HPLC Column at a Flow Rate of 1 ml/min...

Based on these results, what two amino acids are present in your unknown dipeptide As shown in Table 6-1, it may be difficult to resolve some PTC-amino acids (PTC-arginine and PTC-threonine). Still, this mobile-phase gradient will resolve the majority of the PTC-amino acid derivatives. How might the conditions of this experiment be changed to resolve molecules such as PTC-arginine and PTC-threonine ... 

The retention times for each of the PTC-amino acids described above (Table 6-1) will not apply to a chromatography experiment done with this mobile-phase system. If this alternative gradient is used, these values will have to be determined independently. 

Separation protocols System 420A hydrolyzer and derivatizer utilizing a PTC-amino acid pre-column derivatization reaction on-line with a 130A analyzer system and a 920A data collection system (PE/ABI). Column 2.1 x 250 mm ODS PTC column (PE/ABI). Tables I and II describe the conditions for hydrolysates and physiological samples, respectively. 

Figure 1 Structures of PTC-Amino Acids used during this study.

Figure 3. Elution pattern of the two PTC-amino acid separations showing the retention shifts for the indicated derivatized amino acids. A. Standard conditions B. Physiological conditions...

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 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 a L-proline-cupric acetate additive. Chiral reagents, such as Marphey s reagent and OPA/IBLC (A-isobutiril-L cysteine), were successfully used for racemization analysis within 80 min. 

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. 

An even simpler protocol is employed in the formation of A-phenylthiocar-bamoyl derivatives (PTC-amino acids, C6H5NHCSNHCHRC02H Figure 4.1) by reaction in a suitable buffer with the Edman reagent, phenyl isothiocyanate (West and Crabb, 1989). 

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. ... 

Figure 13.20 HPLC of amino acid derivatives detected by 254 nm UV absorption (a) 200 pmol of PTC-amino acid standard, including pbospboserine (PH-S), pbospbothreonine (PH-T), bydroxy-proline (OH-P), galactosamine (Gal), norleucine (NLE, 1 nmol internal standard), excess reagent (Re), and other amino acids designated by one letter codes listed in Fig. 13.19 and (b) Analysis of a human fingerprint, taken up from a watchglass using a mixture of water and ethanol. (Courtesy of National Gallery of Art and the Andrew W. Mellon Foundation.) (Cazes, used with permission.)...

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. 

The use of the o-amino-PTC-amino acid structure (1) in modelling studies using molecular graphics and superimposition on thiorphan as a reference structure, led to the design of 4-carboxymethylamino-4-oxo-3-(2 -amino-... 

In normal-phase chromatography the 2D technique was used to resolve two mixtures of 10 and 11 PTC-amino acids, by eluting with ethanol-chloroform (2 1 v/v) in the first direction and methanol-dioxane-chloroform (1 1 1 v/ v/v) in the second direction. 

In reversed-phase chromatography, the separations of three mixtures of seven PTC- amino acids were effected by eluting with acetonitrile-4% sodium azide - - 2% starch solution (pH = 6.5) (2 8 v/v). 

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). 

Amino acid (0.5-1.0 g) is added to aqueous pyridine (1 1,25 ml) in a stoppered tube. The solution is adjusted to pH 9.0 with IN NaOH and placed in a water bath at 40°C. Phenyl isothiocyanate (1.2 ml) is added with shaking during a reaction time of about 30 min. Additional alkali is added to maintain the pH at 9. The mixture is extracted repeatedly with benzene to remove excess reagent and pyridine. When there is no further uptake of alkali, a slight excess of IN HCl is added to precipitate the PTC-amino acid. The mixture is filtered and warmed with HCl (IN, 30 ml) at 40 C for 2 h. The PTH-derivative crystallizes upon cooling, and further yields are obtained by concentration of mother liquors. Most of the derivatives are recrystallized from aqueous acetic acid or ethanol. 

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). 

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