ABSTRACT
Aggregated γ-75k secalins were isolated by preparative reversed-phase (RP) HPLC of the prolamin fraction of rye flour Danko and were digested with thermolysin. The resulting peptides were preseparated by gel permeation HPLC into eight fractions (G1–G8). Peptides that were linked by disulfide bonds (cystine peptides) were identified by means of differential chromatography (RP-HPLC before and after the reduction of disulfide bonds). The cystine peptides present in fractions G3–G7 were isolated by preparative RP-HPLC and characterized by sequence analysis and, in parts, by mass spectrometry. Accordingly, the eight cysteine residues of the C-terminal domain of γ-75k secalins were linked in the same positions as the intramolecular disulfide bonds of γ-gliadins of wheat. The cysteine residue located at position 12 of the N-terminal domain and characteristic for γ-75k secalins was linked by an intermolecular disulfide bond with a corresponding residue of the same protein type. This cysteine residue is likely to be responsible for the aggregative nature of γ-75k secalins similar to a cysteine residue in the N-terminal domain of LMW subunits of wheat glutenin. In contrast to LMW subunits of glutenin, γ-75k secalins do not possess an additional cysteine residue in the C-terminal domain that forms a second intermolecular disulfide bond. Therefore, the polymerization of γ-75k secalins is limited and the formation of large gluten-like aggregates of rye storage proteins is restricted.
