THE LOCATION OF DISULFIDE BONDS IN ALPHA-TYPE GLIADINS

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two alpha-type gliadins (alpha 3- and alpha 8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from alpha 3-gliadin and three cystine peptides from alpha 8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both alpha-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from alpha 8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of alpha-type gliadins described in the literature, the cystine peptides from alpha 3- and alpha 8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively(11). The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of alpha-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined far low M(r) subunits of glutenin and glutenin-bound gamma-type gliadins(6). Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed. (C) 1995 Academic Press Limited