Purification and Characterization of Ara h1 and Ara h3 from FourPeanut Market Types Revealed Higher Order Oligomeric Structures (1)
Ara h1 is recognized by >90% of the peanut-hypersensitive individuals. Ara h3 has been identified as a major or minor allergen, depending on the population sampled. This allergen triggers an IgE-mediated reaction in 44−77% of the peanut allergic population. The purification of native Ara h3 as a heteromultimeric protein indicated a molecular weight of ∼400 kDa. The fractionation by anion exchange chromatography of the same allergen shows a number of peptides with molecular weights ranging from 14 to 45 kDa by SDS-PAGE.
The Virginia type is mainly used for roasted and salted peanuts in shell and shelled and honey-coated snacks. The Spanish type is typically used in peanut butter industries, in shelled roasted peanuts with or without testae, and in confectionery factories. The Valencia type is usually consumed in-shell after roasting and boiling, whereas the Runner type is utilized in peanut butter manufacture, in salted nut snacks, and in confectionery.
A High Q column (Bio-Rad) separated Ara h1 and Ara h3 from the protein extract of Middleton with a linear salt gradient of 0−0.4 M followed by a rapid increase to 1 M and maintained for 100 min. Ara h3 bound more strongly to the solid phase than Ara h1 and needed a higher salt concentration to elute. The acidic (∼36, ∼38, ∼42 kDa) and basic (∼23 kDa) subunits of Ara h3 were eluted in fractions 2 and 3 at 0.34−0.36 and 0.44−1.0 M NaCl, respectively.
The apparent molecular weight of Ara h1, as calculated by the protein markers on the size exclusion chromatography, varied between ∼430 and ∼550 kDa, suggesting that the native Ara h1 may exhibit an oligomeric structure rather than a trimeric conformation (∼180 kDa).
The differential distribution of Ara h3 trimer and hexamer may be due to the differences between post-translational modification of 11S globulin storage proteins and stability of this conformation in the experimental conditions (i.e., 0.15 M NaCl). The SDS-PAGE of the two major peaks of Menzies and Walter showed two major protein bands with molecular weights of ∼42 and ∼38 kDa, whereas Middleton and Kelinci had three major protein bands of acidic subunits at ∼42, ∼38, and ∼36 kDa, respectively.
The two acidic subunit bands (∼38 and ∼42 kDa) of Menzies cultivar migrated to pI values ∼5. The third acidic subunit (∼36 kDa) was slightly more acidic than the other two subunits and migrated to a pI lower than ∼5.
Protein spots 3−28 were identified by the MS analysis and are listed.
The antibodies raised against Ara h1 from Middleton were highly specific to Ara h1, showing no detectable nonspecific binding with other peanut proteins. The antibodies raised against Ara h3 from Middleton demonstrated specific binding to all three subunits of Ara h3.
The purified Ara h3 from the same cultivars/market types, however, exhibited different quaternary structures, showing differential distribution of trimeric and hexameric structures.