Disulphide 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 ethanol. The insoluble portion (whole glutenin) was partially hydrolysed with trypsin at pH 6.5 and separated on a Sephadex G25 column. The high molecular weight fraction 1 was further hydrolysed with pepsin at pH 2.0. To remove low molecular weight proteins, a portion of whole glutenin was extracted with dilute acetic acid. The residue (enriched glutenin), which contained mostly LMW and HMW subunits of glutenin, was hydrolysed with thermolysin at pH 6.5. The peptic and tryptic hydrolysates were separated on a Sephadex G25 column and the peptide fractions with the highest cystine content were separated further by reversed-phase high-performance liquid chromatography (RP-HPLC). Cystine peptides were detected by differential chromatography (RP-HPLC prior to and after 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 characterized and assigned to known sequences of gluten proteins; 16 peptides confirmed the positions of disulphide bonds present in LMW subunits and gamma-gliadins, as described previously. For the first time, a cystine peptide has been isolated, representing an intermolecular disulphide bond between the y-type of HMW and LMW subunits. Furthermore, a cystine peptide was assigned to gamma-gliadins; thus, all cysteine residues of gamma-gliadins are documented by at least one cystine peptide. One peptide analysed came from the alpha-amylase inhibitor CM 16. Altogether the results indicate that the intramolecular linkages of gluten proteins are not formed at random, but are strongly directed.(ABSTRACT TRUNCATED AT 250 WORDS)
Keck
B
B
Deutsche Forschungsanstalt für Lebensmittelchemie und Kurt-Hess-Institut für Mehl- und Eiweissforschung, Garching, Germany.
Köhler
P
P
Wieser
H
H
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003553:Cystine; D004220:Disulfides / Q000032:analysis; D005983:Glutens / Q000737:chemistry*; D008969:Molecular Sequence Data; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D013820:Thermolysin; D014908:Triticum
D004220:Disulfides; D010446:Peptide Fragments; D003553:Cystine; D005983:Glutens; D010434:Pepsin A; D013820:Thermolysin