Kinetic Analysis of Pepsin Digestion of Chicken Egg White Ovomucoid and Allergenic Potential of Pepsin Fragments

Abstract

Background: The allergenic potential of chicken egg white ovomucoid (OVM) is thought to depend on its stability to heat treatment and digestion. Pepsin-digested fragments have been speculated to continue to exert an allergenic potential. OVM was digested in simulated gastric fluid (SGF) to examine the reactivity of the resulting fragments to IgE in sera from allergic patients. Methods: OVM was digested in SGF and subjected to SDS-PAGE. The detected fragments were then subjected to N-terminal sequencing and liquid chromatography/mass spectrometry/mass spectrometry analysis to confirm the cleavage sites and partial amino acid sequences. The reactivity of the fragments to IgE antibodies in serum samples from patients allergic to egg white was then determined using Western blotting (n = 24). Results: The rate of OVM digestion depended on the pepsin/OVM ratio in the SGF. OVM was first cleaved near the end of the first domain, and the resulting fragments were then further digested into smaller fragments. In the Western blot analysis, 93% of the OVM-reactive sera also bound to the 23.5- to 28.5-kDa fragments, and 21% reacted with the smaller 7- and 4.5-kDa fragments. Conclusion: When the digestion of OVM in SGF was kinetically analyzed, 21% of the examined patients retained their IgE-binding capacity to the small 4.5-kDa fragment. Patients with a positive reaction to this small peptide fragment were thought to be unlikely to outgrow their egg white allergy. The combination of SGF-digestibility studies and human IgE-binding experiments seems to be useful for the elucidation and diagnosis of the allergenic potential of OVM.