Structural Effection And Identification Of Modification Sites Of Shrimp (Metapenaeus Ensis) Tropomyosin By Carbonylation

With the development of modern society, tremendous changes have taken place in the diet structure of humans. And the incidence of allergic diseases that are related to food keep increasing year by year, posing a great threat to our lives and health. Shrimp products are crustacean seafood with relatively higher value in China, which are loved by consumers due to its rich nutrition and delicious taste. However, with the shrimp products being more widely applied to the food industries, many consumers suffer from allergy symptoms. This paper focused on the structure of shrimp (Metapenaeus ensis) tropomyosin through comprehensive study, and dicussed the effect of structural changes and modification sites due to carbonylation. Further analysis was made from the view of mechanism in order to provide theoretical guidance for the changes of nature of the allergen. The main contents are as follows:In order to explore the changes of shrimp allergen structure during storage, this paper regarded Metapenaeus ensis as research object. Applyed the unsaturated fatty acids peroxidation product---malondialdehyde (MDA) to oxidize shrimp allergen (tropomyosin) and detected the laws of structural changes. The results showed that tropomyosin crosslinked after oxidation, and four new bands with 2,3,4,5 times of molecular weight by polyacrylamide gel electrophoresis (SDS-PAGE) were observed. Tropomyosin's peak was 88.80? before oxidation, while tropomyosin's peak was 86.14? after oxidation. And the thermo-stability of tropomyosinhas changed slightly after oxidation. Circular dichroism detected that a-helix content decreased for 13.5% and new chemical bonds formed, accompanied with destruction of the secondary and tertiary structure after oxidation. Absorption peak was detected by infrared radiation in 3010.06cm1 which indicated the existence of olefin. It explained that MDA can combine with the active side chains of certain amino acids after oxidation, forming the new carbonyl and making other features to change. Shrimp tropomyosin is composed of 284 amino acids. We identified the oxidative spot of the amino acid by LC-MS-MS, and observed that 4 lysine (76,168,189 and 233),5 glutamine (61,70, 118,147 and 247) and 4 (17,107,203 and 215) were oxidative modified. In the database detection, we found some amino acids were easily modified by malondialdehyde. Especially, methionine was easily oxidated by active oxygen. The oxidation of malondialdehyde could change the structure of tropomyosin.Another peroxidation product of unsaturated fatty acid is 4-hydroxy-nonenal (HNE) oxidized shrimp tropomyosin. Polyacrylamide gel electrophoresis (SDS-PAGE) was observed to exhibit substantially similar bands of tropomyosin before and after the oxidation, indicating that tropomyosin with HNE did not occur to crosslink after oxidative modification. This might be probably due to HNE containing a reactive aldehyde group of small molecules. However, the crosslinking of MDA previously might be a small molecule with two active aldehydes. Since the intensity of ultraviolet absorption of tropomyosin increased and a slight blue shift has occurred after oxidation, the peptide bond might be destroyed. The peak of tropomyosin was 88.80? before oxidation and 59.99? after oxidation. The thermal stability changed obviously, and oxidized tropomyosin came about a significant denaturation with its conformational structure changed. Circular dichroism detected that a-helix content had a reduction of 2.5%, and Van der Waals force of Tropomyosin binding was destroyed, along with the second and tertiary structure change. NMR analysis detected that amino compounds and methyl both changed before and after oxidation. Oxidation sites of amino acids were identified by LC-MS-MS, detecting five alanine (36,77,81,86 and 242),9-leucine (53,57,78,88,94,95,99,113 and 169), three lysine (38,48 and 66), and a histidine (44) with oxidized modification. Similarly, except the discovery that partial amino acid was easily modified with HNE during the database detection,, methionine was also susceptible to oxidation with reactive oxygen species. HNE oxidation caused tropomyosin structural changes.Shrimp processing can have an effect on the structure and properties of shrimp allergen, thereby changing its allergenic activity. In this study, ribose was selected to react with shrimp allergens through glycosylation. The result of SDS-PAGE showed that Maillard reaction increased molecular weight of shrimp allergen. The peak of tropomyosin was 88.80? before oxidation and 35.71? after oxidation, and the thermal stability changed significantly. Circular dichroism detected that the content of a-helix has the reduction of 1.4%. Ribose and tropomyosin binding van der Waals forces were destroyed, with the change in the secondary and tertiary structure. After oxidation sites of amino acids were identified by LC-MS-MS,1 phenylalanine (278), 3 isoleucines (69,92 and 190),5 methionines that were ribose modified (11,50,126, 141 and 171), and 5 methionines that were active oxygen modified (19,50,126,141 and 171) were also detected being modified by oxidation. In the database detection, some amino acids were easily getting oxidative modified by Ribose, in which methionine was also easily modified by active oxygen. Oxidation could cause the structure change in tropomyosin.The experimental results showed that hydroxylated modification may induce changes in the structure of the allergen protein, and modified sites of amino acid. The changes of protein conformation and sites' modification, such as exposed or buried amino acid into intramolecule, may explain the change in the antigenicity of allergens. This research will contribute to the understanding and control of allergens, which is expected to develop hypoallergenic shellfish food and give support to improving the safety of shellfish food.