ABSTRACT
The prolamin fraction of the rye cultivar Danko was reduced with dithioerythritol and separated by reversed-phase HPLC on C18 silica gel. Two major γ-75k secalins, P1 and P2, were collected, purified by rechromatography, derivatized with 4-vinylpyridine, and digested in parallel with α-chymotrypsin, thermolysin, and trypsin. The different enzymatic hydrolyzates were preparatively separated by two-step reversed-phase HPLC on C18 silica gel, and the resulting peptides were characterized by sequence analysis and, in parts, by mass spectrometry. By means of overlapping peptides and by comparison with a known DNA sequence of a γ-75k secalin (gSec2A) derived from a wheat translocation line (2RS. 2BL), 84% of the P1 sequence and 35% of the P2 sequence could be assigned. Heterogeneity at several sequence positions demonstrated that both protein preparations were not pure and contained at least two or three components. The sequence of the C-terminal domain of P1 was almost completely determined except for one of the 148 residues which could not be identified. The partially determined sequences of P2 were highly homologous with those of P1. The results revealed a close relationship between P1, P2, and gSec2A and a high degree of homology with γ-gliadins of wheat including eight cysteine residues in homologous positions. The partially sequenced N-terminal domain of P1 was similar to that of gSec2A and consisted of repetitive sequences rich in glutamine, proline, and aromatic amino acids. Differences from γ-gliadins were found in the strongly increased number of residues, in the more frequent modifications of the repetitive motifs, and in the presence of a cysteine residue at position 12. The partial amino acid sequence of the N-terminal domain of P2 was in agreement with that of P1, besides a few exceptions in single positions and in the presence of a second cysteine residue.
