Many legumin-like seed proteins, although known to cause human allergies, remain poorly defined- especially with respect to their molecular properties, including immunoreactivity. The legumin-like proteins are complex oligomeric proteins and are major storage proteins in several tree nut seeds including almond, cashew, pecan, and walnut. These legumin-like proteins retain immunoreactivity even after subjecting the seeds to a variety of food processing treatments, and therefore may serve as useful markers when developing detection assays to determine presence of trace quantities of the corresponding tree nut residues in food and feed. The current investigation focused on three specific aims: (i) to purify legumin-like proteins from almond, cashew nut, walnut, pistachio and hazelnut seeds, (ii) to characterize structural properties of tree nut legumins in purified, native states, and (iii) to investigate stability of purified, native legumin allergens subjected to select chemical treatments. Amandin and anacardein were purified according to the methods established by Sathe et al., 2002 and Sathe et al., 1997 respectively. Purification of legumin-like proteins from walnut, pistachio, and hazelnuts was attempted by chromatographic techniques. Polypeptide composition of legumin preparations was determined using SDS PAGE and 2D PAGE under reducing conditions. Two-dimensional PAGE was used to investigate presence of isoforms. Secondary structures of purified legumins were estimated by far- UV Circular Dichroic (CD) spectroscopy while fluorescence spectroscopy was used to study tertiary structure. Effects of chemical treatments on protein structures and on immunoreactivity of the legumins were assessed. Chromatographically purified amandin and anacardein preparations contained three proteins each, constituting over 90% of the protein. Two of these three proteins were 11S globulin isoforms as judged by their closely matching polypeptide composition. The third protein, in both amandin and anacardein, exhibited polypeptide composition and properties similar to vicilin-like proteins. When amandin and anacardein preparations were subjected to chemical treatments and studied with CD spectroscopy, SDS treatments caused higher a-helical structures and perturbation in the surface structure in amandin and anacardein. Gradual loss in specific mAb immunoreactivity was detected with increasing concentration of SDS treatments of amandin and anacardein. Specific mAb immunoreactivity appeared to be influenced by location of mAb-reactive peptide stretches on amandin and anacardein structures generated by homology modeling. GuHCl treatment resulted in disruption of amandin and anacardein structures. The loss of secondary and surface structure was more gradual (0 to 2M GuHCl) in amandin than in anacardein (>0.2 M GuHCl), perhaps owing to higher random coil structure in amandin. Specific mAb immunoreactivity of GuHCltreated amandin and anacardein did not change significantly compared to their corresponding controls. Amandin conformational structure in up to 2 M urea appeared to alter only marginally compared to amandin control. More significant changes were observed in anacardein when treated with urea: major loss of a-helical structure in >0.2 M urea, and gradual disruption of surface structure at higher urea concentrations. Specific mAb immunoreactivity of both amandin and anacardein in 2.5 M urea did not change significantly relative to the corresponding controls. Reduction (bME) and heat denaturation of amandin and anacardein resulted in fractional loss of surface structure, and significant loss of immunoreactivity in 3 of the 4 mAbs used for probing the proteins.