In Vitro Susceptibility of Oxidized Myosin by μ-Calpain or Caspase-3 and the Determination of the Oxidation Sites of Myosin Heavy Chains

The effect of susceptibility to in vitro oxidation on the degradation of myosin isolated from beef muscles via mu-calpain or caspase-3 was examined, and the measurement of the oxidation sites of myosin heavy chains was performed. Myosin was incubated with hydroxyl free radical-generating systems, which were composed of 0.01 M FeCl3, 0.1 M ascorbic acid, and 0, 25, 50, and 100 mu M H2O2 at 37 degrees C for 20 min. The oxidized myosin then reacted with mu-calpain or caspase-3 at 37 degrees C for 30 min, respectively. The results showed that protein oxidation systems in vitro resulted in different levels of myosin oxidation, leading to significant changes in the secondary structure of myosin (P < 0.05). The sodium dodecyl dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting results showed that in vitro oxidation promoted myosin degradation via pcalpain or caspase-3. Proteomics research suggested that the number of myosin oxidation sites increased constantly with the increase of oxidation levels. Oxidation sites of myosin were mainly cysteine, methionine, arginine, histidine, tyrosine, lysine, and asparagine. These results indicated that oxidation using H2O2 in the range of 0-100 mu M could increase the degradation of myosin via p-calpain and caspase-3 due to increased exposure of the oxidation sites of myosin.