Separation of the Tryptic Peptides and Cyanogen Bromide Fragments of the Human Embryonic Zeta (§) Chains of Hemoglobin Portland I and II by Reverse Phase High Performance Liquid Chromatography

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Abstract
The amino acid compositions of tryptic peptides and cyanogen bromide fragments of the purified .zeta. chain of Hb Portland I (.zeta.2YL2) and Hb Portland II (.zeta.2.beta.2) were determined. The Hb were obtained from blood from neonates with hydrops fetalis due to homozygous .alpha.-thalassemia. The globin chains, tryptic peptides and cyanogen bromide fragments were all separated by reverse phase high performance liquid chromatography (HPLC). Several different types of C-18 columns were used with 2 different developer systems. The tryptic peptides of aminoethylated .zeta. chain were separated using an ammonium acetate-acetonitrile gradient. An aqueous trifluoroacetic acid-1-propanol developer gradient was used for the separation of cyanogen bromide fragments. Of the 17 tryptic peptides obtained, 2 (.zeta.T10a and .zeta.T10b) resulted from the unusual cleavage of a Tyr-Ile peptide bond. This was observed even when using TPCK treated trypsin. Trypsin apparently will hydrolyze the Tyr-X bond provided either Ala or Ile is bonded to the N-terminal side of Tyr and Ile, Leu, or Gly is bonded to the C-terminal side of the Tyr residue.