DISULFIDE BONDS IN WHEAT GLUTEN - CYSTINE PEPTIDES DERIVED FROM GLUTEN PROTEINS FOLLOWING PEPTIC AND THERMOLYTIC DIGESTION

Gluten from the wheat variety Rektor was extracted with 70% aqueous et hanol. The insoluble portion (whole glutenin) was partially hydrolysed with trypsin at pH 6.5 and separated on a Sephadex G25 column. The hi gh molecular weight fraction 1 was further hydrolysed with pepsin at p H 2.0. To remove low molecular weight proteins, a portion of whole glu tenin was extracted with dilute acetic acid. The residue (enriched glu tenin), which contained mostly LMW and HMW subunits of glutenin, was h ydrolysed with thermolysin at pH 6.5. The peptic and tryptic hydrolysa tes were separated on a Sephadex G25 column and the peptide fractions with the highest cystine content were separated further by reversed-ph ase high-performance liquid chromatography (RP-HPLC). Cystine peptides were detected by differential chromatography (RP-HPLC prior to and af ter reduction of disulphide bonds) and then isolated by preparative RP -HPLC. After reduction, cysteine peptides were alkylated and analysed for their amino acid sequence. Altogether, 19 cystine peptides were ch aracterized and assigned to known sequences of gluten proteins; 16 pep tides confirmed the positions of disulphide bonds present in LMW subun its and gamma-gliadins, as described previously. For the first time, a cystine peptide has been isolated, representing an intermolecular dis ulphide bond between the y-type of HMW and LMW subunits. Furthermore, a cystine peptide was assigned to gamma-gliadins; thus, all cysteine r esidues of gamma-gliadins are documented by at least one cystine pepti de. One peptide analysed came from the alpha-amylase inhibitor CM 16. Altogether the results indicate that the intramolecular linkages of gl uten proteins are not formed at random, but are strongly directed. For intermolecular linkages, however, different combinations were found.