PTH-amino acid

C. Mass Spectra of TBDMS Derivatives of PTH-Amino Acids... 

Protein Sequencing. The xylanase protein sequence was obtained by stepwise automated E an degradation ter an initial double-coupling protocol, using an Applied Biosystems model 470A gas-phase sequenator equipped with an on-line Applied Biosystems model 120A PTH-amino acid analyzer 13). 

The chemistry of this reaction has been extensively studied by Use and Edman. Although the reaction velocity is different for various amino acids, the reaction is complete in 1 N HCl at 80 °C within 10 min, with virtually all amino acids. This treatment, however, would cause partial hydrolysis of the remaining peptide. The method was made amenable to automation when Edman suggested to separate the thiazolinone derivative from the remaining peptide by extraction of the liberated thiazolinone derivative into butyl chloride and to perform the conversion into the PTH-amino acid outside the reaction vessel. 

Originally, Edman hydrolyzed the PTH-amino acids and identified the free amino acids by paper chromatography 0. Since not all of the amino acids are sufficiently resistant to this treatment Edman and Soquist subsequently devised methods for the direct identification of the PTH-amino acids. Paper chromatography has now generally been abandoned in favour of the more sensitive thin layer chromatography Recently, gas-liquid chromatography... 

The unstable thiazolinones are converted into stable hydantoines in order to facilitate their identification. Conversion and identification are carried out outside the instrument after extraction of the thiazolinones with butylchlor-ide. The conversion reaction as well as the problems associated with identification of the PTH-amino acids were studied in detail by Edman and described explicitly in Needleman s book on Protein Sequence Determination Conversion is generally carried out in 1 N HCl at 80 °C within 10 min. The PTH-derivatives are extracted from the aqueous phase with ethyl acetate with the exception of PTH-arginine, PTH-histidine and PTH-cysteine which remain in the aqueous phase. 

Fig. 7a and b. Gas chromatograms of PTH amino acids, a) Apolar amino acids b) silylated polar amino acids. 5 n Mol of each PTH-amino acid were applied. Chart speed 0.5 /min., gas chromatograph GC-45 , Beckman Instruments... 

In view of its rapidity we found thin layer chromatography convenient for identification of the amino acids liberated by the first 20—30 degradation cycles. For identification of PTH-derivatives from additional degradation steps we prefer gas-liquid chromatography because of its merits mentioned above, particularly its greater sensitivity. Several colorimetric reactions and chromatographic systems are available for the identification of those PTH-amino acids which remain in the aqueous phase when the PTH-derivatives are extracted with ethyl acetate 23.24,25,13) our hands, thin layer electrophoresis was found to be satisfactory 26,27)... 

R-P separation of PTH-amino acids 4 gradient parameters and flow rate 77)... 

Bhown, A.S., J.E. Mole, and J.C. Bennett, improved procedure for high-sensitivity microsequencing use of aminoethyl amino-propyl glass beads in the Beckman sequencer and the ultrasphere ODS column for PTH amino acid identification. Anal Biochem, 1981.110(2) 355-9. 

Palladium-calcein reaction with PTH-amino acids.159... 

The formation of methyl- (MTH) or phenylthiohydantoin (PTH) amino acids is a valuable technique for sequencing of amino acids in peptides and proteins by the Edman degradation procedure [1], HPLC is very useful for the separation of MTH- or PTH-amino acids as adsorption [2,3], reversed-phase [4] and ion-exchange [S] chromatography. 

PITC). The mixture is warmed at 40 °C for 1 h, then diluted with 1 ml of water and the excess of MITC (or PITC) is removed by extracting four times with 2-ml volumes of benzene. The aqueous layer is evaporated and dried in a vacuum desiccator over sodium hydroxide. The general scheme for the formation of PTH-amino acids is illustrated in Fig.4.1. For peptide hydrolysis, 1.5 ml of a mixture consisting of equal volumes of 3 N hydrochloric acid and 60% acetic acid are added and the reaction is incubated in a nitrogen atmosphere for 30 min at 40 °C. The mixture is diluted with 2 ml of water and the thiohydantoin derivative is extracted with 2 ml of ethyl acetate followed by 2 ml of benzene. The combined extracts are used for chromatography. 

PTH-amino acids may be separated with a liquid-solid adsorption system consisting of small-particle silica gel (particle diameter, 5 pm) in a 50-cm column eluted with methylene chloride-dimethyl sulfoxide-rerr.-butanol in various ratios. The separation of a number of PTH-amino acids on Merkosorb SI-60 (5 pm) with methylene chloride-dimethyl sulfoxide-rerr.-butanol (125 0.1 1.0) is illustrated in Fig.4.3. Absorption is measured at 260 nm. Amounts of less than 1 nmole of each amino acid derivative can be... 

Reversed-phase separation [6] of polar and non-polar PTH-amino acids may be accomplished using Corasil-Cig bonded phase packing (particle diameter, 37-50 jum) and eluting with water-acetonitrile-isopropanol (100 1.5 1). An example of the reversed-phase separation of sixteen PTH-amino acids is given in Fig.4.4. The limit of detection of... 

Fig.4.3. Separation of some PTH-amino acids. Pressure, 250 bar flow-rate, 1.65 ml/min (other details as in text). (From ref. 2 with permission of Pergamon Press, Elmsford, N.Y.)...

The use of ligand exchange has been examined for the analysis of PTH (phenylthio-hydantoin) amino acids separated on silica gel plates [92]. The method is an extension of the procedure developed for organophosphate pesticides [84]. The chromatoplate is sprayed with a solution of palladium(II) chloride and calcein. Palladium complexes with calcein to form a non-fluorescent chelate. However, in the presence of many sulfur-containing compounds, such as PTH-amino acids, the palladium is displaced from the complex liberating free calcein which gives an intense fluorescence. This method is capable of determining 0.1-nmole amounts of PTH-amino acids. 

Concentrations range from 0.01 to 2 % in the modifier (typically 0.005 % in the complete mobile phase). Hydroxy-benzoic acids (2), polycarboxylic acids (3), benzylamines (4), PTH-amino acids (5) and many other families of polar solutes only elute when an appropriate additive is present in the mobile phase. 

The fluorescent beads are manually isolated, washed with 8 M guanidine-HCl and water, and then submitted for sequence analysis. We expect that some of the peptides on the positive beads will be cleaved at the proteolytic site. As a result, the PTH-amino acids detected during each cycle of Edman degradation will have been generated from both of these peptides. This complicates the sequence analysis somewhat. However, it also allows us to determine the proteolytic cleavage site of the peptide, in addition to its uncleaved sequence. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

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