Purification, Biochemical Characterization and Digestive Stability Analysis of the Major Allergen Paramyosin From <i>Haliotis discus hannai</i>

Paramyosin (PM) is a structural protein in invertebrates, playing an important role in maintaining the texture of muscle. Although PM has been identified as a major allergen, its characteristics have not been well investigated. In this study, PM with a molecular mass of 97 kDa was purified to homogeneity from Haliotis discus hannai using ammonium sulfate fractionation and hydroxyapatite column chromatography. Liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis identified 11 peptide fragments with a total of 66 amino acid residues, which were 100% identical to PM from Haliotis discus discus . The sequence of the PM gene, including its complete open reading frame of 2583 bp encoding 860 amino acid residues, was determined by molecular cloning. PM is almost a long‐rod chain protein, and α‐helix is the dominant secondary structure. Simulated gastrointestinal fluids digestion showed that PM is highly tolerant to digestive proteinases. A specific polyclonal antibody against PM was prepared. Western blot analysis revealed that PM was the most abundant in H . discus hannai , followed by Rapana venosa , Argopecten irradians , and Uroteuthis chinensis , while PM could not be detected in shrimp Penaeus merguiensis and freshwater fish Hypophthalmichthys molitrix , strongly suggesting the unique existence in shellfish.