CE study of neuroprotective humanin peptide and its derivatives: Interactions with phosphate, sulphate, alkylsulphonates and sulphated-β-CD

Humanin (HN), Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-IIe-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, recently discovered in the human brain, is an important neuroprotective peptide. Some derivatives of HN show even higher biological activity, for example [G-14]-HN, where Ser at position 14 is replaced with Gly. As structurally related HN peptide derivatives have similar chemical properties, their separation by CE is difficult. In this work, the electrophoretic behaviour of HN derivatives including [G-14]-HN, a tryptophan HN derivative [W-14]-HN, several other HN derivatives and HN fragments was studied. While phosphate buffer was used as the general BGE, the effects of the buffer concentration and various additives were examined, including sulphate, heptane sulphonate, 2-morpholinoethanesulphonic acid N-[tris(hydroxymethyl)methyl]-2-amino-ethane sulphonic acid (TES), sulphated-beta-CD and beta-CD. Separation efficiency of 200 000 theoretical plates was achieved in a BGE of 80 mM phosphate at pH 2.5 where seven out of nine major peaks were partially separated. By investigating the influence of concentration of the interrogated ions on peptides migration, the association between positively charged protonated sites of peptides and various anions was proved. Especially a strong interaction with phosphate, sulphate and sulphonate groups was established. Conditional stability constant of the [Pep(z+), (H2PO4-)(n)](z-n) ion associate (n = 1) for [G-14]-HN equals to log K similar to 1.78.