Abstract
Based on our previous method (6) and improvements made by Summers et al. (8), we developed a tlc-technique for two-dimensional separation of 24 most common PTH 1 1 The following nonstandard abbreviations were used: PTH, phenylthiohydantoin; CM-Cys, S-carboxymethylcysteine; CySO 3H, cysteic acid; Metsf, methionine sulfone; N-Phe, N-phenylthiourea; N,N-DPhe, N,N-diphenylthiourea. -amino acids obtained after manual or automated Edman degradations. Using a fluorescent indicator (8) and double-faced micropolyamide sheets (5 × 5 cm) our method allows the complete identification of all derivatives in about 30 min including His/Arg/CySO 3H 1 and Leu/Ile PTHs not separable by Summers' technique. Offering the same lower limit of detection at 254 nm (0.05–0.2 n m), in contrast to the dansyl-Edman technique, the micropolyamide tlc in addition to direct Edman degradation allows correct placing of Gln and Asn within sequences. We also set up a method for identification in 20–30 min of 10 individual PTH samples on the same 5 × 5-cm sheet by consecutively applying two solvent systems in identical direction. Compared with gc the advantages of this convenient and inexpensive micromethod consist in completeness of separations, increase of sensitivity, and in simultaneous processing of many samples.
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