Extraction and Purification of Myosin and the Effects of Hydroxyl Radical Oxidation on Its Oxidation Sites

In this study, myosin was extracted from fresh mutton using precipitation centrifugation, purified via column chromatography, and concentrated through ammonium sulfate salting-out combined with ultrafiltration centrifugation. The results demonstrated that the improved protein extraction method was more efficient. The optimal loading amount of myosin onto the ion-exchange column was determined to be 20 mg, and the optimal saturation level for ammonium sulfate salting-out stood at 50%. To explore the impacts of hydroxyl radical oxidation on the structure and oxidation sites of myosin, a hydroxyl radical oxidation system was established by regulating the concentration of H₂O₂ (0, 0.5, 1, 5, 10, and 20 mmol/L), which enabled myosin to undergo oxidation to varying degrees. Research findings indicated that with the increase of H₂O₂ concentration, the carbonyl content of myosin increased by 2.376 times, while the contents of total sulfhydryl, free sulfhydryl, ionic bond and hydrogen bond decreased by 37.02%, 53.24%, 50.98% and 50.85%, respectively, upon treatment with 20 mmol/L H₂O₂ compared to the control group (0 mmol/L). In contrast, the disulfide bond content exhibited a tendency of first increasing and then decreasing. It reached its maximum value when the H₂O₂ concentration was 10 mmol/L, which was 2.28 times that of the control group. Measurements using a fully automatic amino acid analyzer revealed that four amino acids, namely Cys, Tyr, Phe, and His, were susceptible to oxidation, with the order of their susceptibility being Cys>Tyr>His>Phe. LC-MS/MS detection showed that under mild oxidation conditions, hydroxyl radicals mainly attacked the proteins around the SH1-SH2 region. When subjected to severe oxidation at an H₂O₂ concentration of 20 mmol/L, a large amount of myosin aggregated, and these aggregations primarily occurred in the head helical region and the 1502~1783 region of myosin.