Food Science and Technology Department
Date of this Version
The identification and characterization of soluble pecan proteins was found to be qualitatively dependent on the chemical characteristics of the extraction buffer that also had a significant influence on the total protein content of an extract. The ionic strength of the extraction buffer had a greater impact on protein extractability than pH. A high salt phosphate buffer (0.01 M PBS, 1M NaCl, pH 7.2) was used as an optimal extraction solution that allowed remarkable detection of clinically relevant allergens.
The 2S albumin family of seed storage proteins has been shown to be one of the most stable allergens, thus potentially making these proteins clinically relevant for allergic sensitization. In this study, we sought to purify native 2S albumin from pecan to further characterize this putative allergen. The protocol for purification and isolation of 2S albumin protein from pecan, Car i 1, was developed employing two size-exclusion chromatography steps. Sequence identification of pecan proteins was done by means of LC-MS/MS ion trap mass spectrometry.
The soluble high molecular weight pecan proteins were rapidly digested by pepsin in simulated gastric fluid (SGF) whereas the low molecular weight proteins were more stable or were reduced to digestion resistant proteins and polypeptides. The purified 2S albumin, Car i 1, from pecan was found to be resistant to digestion in SGF and comparatively stable to proteolysis by trypsin and pancreatin in simulated intestinal fluid (SIF).
Digestion of purified Car i 1 in SGF and SIF resulted in formation of different digestion-resistant peptides, which included important epitopes that were retained despite extensive in vitro digestion. These peptides were capable of binding IgE antibodies from allergic individuals. Digestion stability of Car i 1and formation of stable, digestionresistant antigenic polypeptides may contribute to allergic sensitization to pecans in susceptible individuals.
Advisor: Joseph L. Baumert
A Thesis presented to the faculty of the graduate college at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science. Major: Food Science & Technology. Under the Supervision of Professor Joseph L. Baumert. Lincoln, Nebraska: December, 2011.
Copyright 2011 Jelena Spiric.