Application of Edman's Phenylisothiocyanate Method in the Determination of C-terminal Amino Acids and Peptides after Hydrazinolysis.

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Abstract
Edman''s phenylisothiocyanate method has been used for identification and quantitative determination of the C-terminal amino acids released in hydrazinolysis of fibrinopeptides A and B and of insulin. The method is found to be particularly suitable in cases where relatively large amounts of C-terminal peptides are formed, since it can also be used for sequence determinations of these peptides. For more detailed studies of complicated hydrazinolysates, Edman''s method can be preceded by electrophoretic separation. The C-terminal sequences of fibrinopeptides A and B and of insulin as found in the present work are in accordance with the known structure of these substances. Certain peptide bonds are found to be highly resistant to hydrazine under the conditions used (100[degree]C for 10 h). In hydrazinolysis of fibrinopeptide A, only an inappreciable quantity (5%) of ornithine is obtained from the C-terminal amino acid arginine, whereas a good yield (about 70%) is obtained from the di-and tripeptides val-orn and gly-val-orn, respectively, deriving from the C-terminal end, Thus, the peptide bond val-orn (val-arg) seems to be only inappreciably split in hydrazinolysis. The C-terminal peptide bond in fibrinopeptide B, ala-orn (ala-arg), also seems to be relatively resistant, as is the penultimate peptide bond in the B-chain of insulin, pro-lys. The phenylthiohydantoin ( PTH) derivative of ornithine has been synthesized. A solvent system is described for separation of the phenylthiohydantoin derivatives of lysine and ornithine.
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