Separation of gliadin peptides for investigation of the injurious agent(s) in gluten sensitive enteropathy

Abstract

Crude gliadins isolated from three wheat varieties (Karl, Tam 107 and CS- 93) were subjected to in vitro hydrolysis by the extracellular enzymes pepsin, peptidase and pancreatin. Gliadins from one variety (Karl) also were exposed to the intracellular enzymes cathepsins Band D. A reverse phase high performance liquid chromatography (RP-HPLC) method was developed to separate unhydrolyzed and hydrolyzed gliadins. The unhydrolyzed crude gliadins were resolved into 12-14 peaks, with at least five peaks that appeared to be common to all three wheat varieties. Gliadin peptides were resolved into between 44 and 71 peaks, suggesting that a large numbers of peptides are derived from proteins present in more than one of the four major gliadin fractions (i.e.; α, β, γ, and Ï - gliadins). No major differences were detected between chromatograms of extracellular digests and those of extracellular /intracellular digests indicating that cathepsins Band D may not contribute to more complete gliadin digestion. The molecular weights and amino acid sequences of gliadin peptides will need to be determined by HPLC mass spectrometry (HPLC-MS) for accurate qualitative and/or quantitative comparisons of individual digests. Our in vitro hydrolysis/RP-HPLC methods may be applicable, however, in the generation of celiac active peptides for future toxicity testing.