DOI: 10.1094/CC-82-0541 |  VIEW ARTICLE

Studies of Partial Amino Acid Sequences of gamma-40k Secalins of Rye.

Claudia Gellrich (1), Peter Schieberle (1), and Herbert Wieser (1,2). (1) Deutsche Forschungsanstalt für Lebensmittelchemie und Hans-Dieter-Belitz-Institut für Mehl und Eiweißforschung, Lichtenbergstraße 4, D-85748 Garching, Gemany. (2) Corresponding author. E-mail: <h.wieser@lrz.tum.de> Cereal Chem. 82(5):541-545. Accepted March 4, 2005. Copyright 2005 AACC International, Inc.

Though gamma-40k secalins are a major protein type within rye storage proteins, total amino acid sequences are not as well known as the gluten proteins of wheat. Well-reputed structural features such as amino acid compositions and molecular masses indicated a close relationship between gamma-40k secalins and gamma-gliadins of wheat, but the degree of homology of amino acid sequences and the positions of intramolecular disulfide bonds are unknown. Therefore, two major components of gamma-40k secalins (R1, R2) were analyzed for partial amino acid sequences. The R1 and R2, derivatized with 4-vinylpyridine, were isolated from the prolamin fraction of rye cultivar Danko by means of a two-step RP-HPLC on C(18) silica gel. The proteins were digested in parallel with trypsin and thermolysin, and the partial hydrolyzates were separated by RP-HPLC. Simultaneous measurement of UV absorbance at 210 and 254 nm allowed the detection of all peptides eluted as well as the specific detection of pyridylethylated cysteine peptides. Isolated peptides were characterized by sequence analysis, and in parts by mass spectrometry, and assigned to known sequences of gamma-gliadins. The results demonstrated that the N-terminal domain of R1 and R2 remained undigested after tryptic hydrolysis; they were in agreement with the N-terminal domain of gamma-gliadins in their molecular masses and in the absence of cysteine residues. Most of the isolated peptides originated from the C-terminal domains, they covered 83% (R1) and 77% (R2), respectively, of the C-terminal domain of a known gamma-gliadin (clone pW1020). Comparison of R1 and R2 revealed differences only in a few sequence positions. The degree of homology between the C-terminal domains present in gamma-40k secalins and gamma-gliadins was approximately 85%. All eight cysteine residues of gamma-gliadins were found in R1 and R2 sequences. Remarkably, sequences close to corresponding cysteine residues were identical for gamma-40k secalins and gamma-gliadins. Therefore, it can be assumed that the positions of intramolecular disulfide links are homologous.