Purification and biochemical characterization of a novel allergenic protein from tatary buckwheat seeds

An allergenic protein from tatary buckwheat seeds, designated TBt, was purified by salting out, anion exchange chromatography and size exclusion chromatography (SEC). Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SEC showed that the allergenic protein was composed of two subunits linked by disulfide bonds, which we named TBa and TBb. TBa, TBb and TBt have estimated molecular weights of 24, 34 and 56 kDa, and isoelectric points of 6.85, 5.85 and 7.00, respectively. The immunological characteristics of the three proteins as analyzed by enzyme-linked immunosorbent assay revealed that TBa and TBb have comporable IgE binding activity, both higher than that of TBt. Determination of sulfhydryl (-SH) and disulfide (-S-S-) groups in TBt showed that most cysteine residues exist as disulfide linkages rather than free -SH groups. We calculated that TBt has an -S-S- content of 1.978 M/M protein, indicating that TBt contains one disulfide bond and could be the precursor of a 24 kDa allergenic protein from tatary buckwheat seeds.