Approximately twenty years ago, Edman introduced the use of phenylisothiocyanate as a reagent for determining amino acid sequences in proteins¹. This reagent reacts only with the alpha and epsilon amino groups of proteins of all the functional groups present to produce the phenylthiocarbamyl derivative of the protein (A), reaction 1.

The phenylthiocarbamyl derivative of the N-terminus is cleaved in strongly acidic, non-aqueous solution at the first peptide bond producing the 2-anilino-5-thiazolinone derivative of the amino acid (B) formerly in the N-terminal position of the protein plus the protein lacking the N-terminal amino acid, reaction 2.

This protein can be separated and treated again with phenylisothiocyanate to identify 2nd, 3rd, 4th, etc., amino acid of the sequence.

The 2-anilino-5-thiazolinone derivative is changed by aqueous acid to the more stable 3-phenyl-2-thiohydantoin (PTH) reaction 3, which can be quantitated in a number of ways.

The 3-phenyl-2-thiohydantoin (PTH) absorbs strongly in the UV at approximately 16,000⁸ and have a minimum absorption at 245 nm. As an index of purity the ratio OD 269/OD 245, which should be about 2.3 - 2.7, can be used for the majority of these derivatives³. Vigorous hydrolysis will convert the derivatives back to the amino acid. With the exception of the derivatives of histidine, arginine and cysteic acid all the PTH derivatives are very soluble in organic solvents and insoluble in water.

The PTH derivative may be quantitated by using the high extinction value in the UV⁸. They may be identified by chromatographic methods, e.g., thin layer ⁴, paper chromatography^4,5, partition chromatography⁶, gas chromatography⁷, silica gel thin layer⁸, polyamide⁹ and glass paper¹.

The PTH derivatives can be detected on chromatography by the use of iodine-azide reaction² or by fluorescent methods¹² or other chemical methods¹³.

The manual techniques have been combined and automated into a protein sequenator¹⁴ which has been used to determine the sequence of several proteins, e.g., 60 amino acid residues in myoglobin¹⁴.

In addition to using the Edman method for sequence purposes from the N-terminus of proteins, other uses of PTH derivatives have been reported.

    1. The activation of trypsinogen by enterokinase has been followed¹⁵.

    2. The conversion of fibrinogen to fibrin¹⁶.

    3. Determination of the proteolytic activity of thrombin¹⁷.

    4. Used in the determination of the C-terminal amino acid after application of Akbori's hydrazinolysis method¹⁸.

A very useful reference to the methodology and application of the PTH derivatives in sequencing a protein can be found in "Protein Sequence Determination" edited by S.B.Needleman, Springer-Verlag, 1970, p.211.

The following PTH derivatives are exceptionally pure having been fully analyzed by mass spectrometry and conforms perfectly to theory.

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