Separation of pure toxic peptides from a β-gliadin subfraction using high-performance liquid chromatography

Abstract The β v subfraction was isolated from peptic-tryptic digests of β-gliadin by chromatography on Biogel P-10 and applied to a Lichrosorb RP-18 or a μ-Bondapak C-18 column. Fractionation was achieved using reverse-phase high-performance liquid chromatography with a linear gradient of acetonitrile in ammonium acetate. A better resolution was obtained with the μ-Bondapak column. The first-eluted peptides a, b and c1 appeared to be well purified and apparently uncontaminated. Analysis of peptides a and b showed that they contained 40 to 42% glutamine/glutamic acid, 20 to 23% proline, 14 to 16% valine and 8 to 10% leucine. They had valine as the N-terminal amino acid and their molecular mass was estimated as 5500 using sodium dodecylsulfate electrophoresis after dansylation. Peptide c1 differed from peptides a and b in containing less valine and leucine and additional amino acids such as threonine, phenylalanine and tyrosine. In addition, it had a lower molecular mass (approximately 5000) and serine as the N-terminal amino acid. Peptide b exhibited an obvious cytotoxicity for cultured cœliac jejunal mucosa at a very low concentration (0.01 g/l) and was the most toxic peptide.