Separation of pure toxic peptides from a beta-gliadin subfraction using high-performance liquid chromatography.
The beta v subfraction was isolated from peptic-tryptic digests of beta-gliadin by chromatography on Biogel P-10 and applied to a Lichrosorb RP-18 or a mu-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 mu-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 coeliac jejunal mucosa at a very low concentration (0.01 g/l) and was the most toxic peptide.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D046508:Culture Techniques; D005903:Gliadin / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000032:analysis; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000302:isolation & purification*; D010940:Plant Proteins / Q000032:analysis*
D000596:Amino Acids; D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; D005903:Gliadin