Identification
of phenylthiohydantoins of amino acids-by thin-layer chromatography
The, identification of amino acids as their phenylthiohydantoins (PTHs) is of major importance in sequence determinations of peptides and proteins by the EDMAN degradaiion technique. The need for a rapid procedure has increased recently because of the desirability of matching the output from the protein sequenator which releases one amino acid derivative from the peptide chain every go mini. EDMAN~ recommends thin-layer chromatography for this purpose and runs ten samples simultaneously on thin layers of silica gel on glass plates, with reference mistures in alternate lanes to assist identification. As a rule, two plates are used, one for chromatography in system H for the resolution of the more polar amino acid derivatives, and one in system D to resolve PTHs with 'very similar mobilities, in particular isoleucine from leucine and valine from phenylalanine.
Likewise, JBPPSON AND SJ~QUIST~ recommend a thin-layer procedure for identification of the PTH-amino acids but use successive solvent systems to effect resolution of all the derivatives. This method is advantageous when the amount of sample is limited because only one plate is required for the identifications. The purpose of this note is to report an improved ED&W 1-I system2 which, in conjunction with ninhydrin colour reactions of the PTH-amino acids, allows identification to be made after only one cliromatograpliic step.
The recommended system is as follows. The ratio of ethylene chloride to acetic acid in the solvent is 60 : 7 (cf. EDMANS, 60: 14). In preference to coated glass plates, the chromatography is carried out on silica gel containing a fluorescent dye (Merck, Kieselgel 6017 254.) on an aluminium plate, which is developed for a distance of "r2.5 cm. The pre-coated aluminium plates give smaller, more discrete spots hut the most important improvement is to the relative Rp values of PTH-isoleucine and PTHleucine. A small hut reproducible difference, equivalent to that in the EDMAN D systemc, is obtained for these derivatives. We have also found the pre-coated aluminium plates to have other advantages over the glass plates prepared in the laboratory; they are more resistant to abrasion, the PTH mixtures eshibit more reproducible RF values across the plate, and the colour reactions are more permanent.
Pig. I illustrates, the results obtained with the modified system and shows the separations in systems D and H of ED,MAN~ for comparison. It shows that PTHisoleucine and PTH-pllenylalanine are resolved from PTH-leucine and PTH-valine, respectively. PTH-aspartic acid, PTH-glutamic acid and PTH-tyrosine show increased separations. Samples from our sequenator generally behave normally in the system but occasionally the non-polar derivatives run slightly slower than the standards. This effect was not observed when fresh PTH standard mixtures were used. We have found that loading the samples in ruled lanes2 gives more compact spots and hence * The original plates 1Kicselgcl I? 254) were found to be unsatisfactory for PTH separations. * * System D: xylene. Plate soaked in formamide-acetone (I : 3) and air-dried.' System I-I: e ctkylene chloride-acetic acid (30: 7). Papcr lining soaked with solvent.
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