Biochemical Characterization of γ-75k Secalins of Rye II. Disulfide Bonds

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.