Protein carbonylation and structural changes in porcine myofibrillar protein exposed to metal ion-H2O2-ascorbate and linoleic acid-lipoxidase oxidizing systems

Abstract

The present study aimed to compare two oxidizing systems commonly present in meat for their influence on protein oxidation patterns, with emphasis on the specific lysine-derived markers for protein carbonylation (α-aminoadipic semialdehyde, AAS; lysinonorlucine, LNL) and their relationships with the common markers for protein oxidation. For this purpose, pork myofibrillar proteins (MFP, 5 mg/mL) were suspended in 0.6 M NaCl (pH 7.5) and incubated at 4 ℃ for 24 h with two oxidizing systems: (1) a metal-catalyzed oxidizing system (MOS: 10 µM FeCl3, 100 µM ascorbic acid, and 0–10 mmol/L H2O2), (2) a linoleic acid – lipoxidase oxidizing system (LOS: 7500 units of lipoxidase/mL, and 0–10 mM linoleic acid). Results showed that the amounts of AAS and LNL in both MOS- and LOS-oxidized MFP was proportional to the oxidant concentrations (H2O2 or linoleic acid), while the formation of total carbonyl and total thiol also exhibited similar oxidant-dose-dependent patterns. Meanwhile, the α-helix contents of MFP declined with oxidant concentrations irrespective of the oxidizing systems. The reducing SDS-PAGE revealed that the myosin heavy chain band started to diminish at high H2O2 concentration (5 and 10 mM) in MOS whereas at low level of linoleic acid (0.5 mM) in LOS. Overall, these results demonstrated both oxidizing systems could be involved in the formation of AAS and LNL, and that the generation of AAS and LNL can be used as reliable markers for protein oxidation, but also might be directly involved in protein structural changes and then contribute to the alternations of protein functionality.