Influence Of Protein Oxidation On Desmin And Calpain During Postmortem Ageing

The postmortem ageing of meat is a important process which various proteins and enzymes were involved in. Muscular tissue contain abundant oxidation stimulation factors, therefore oxidation exists during meat ageing, storage and processing. Fat oxidation has been researched widely, but study about protein oxidation is less. This thesis mainly investigated the influence of protein oxidation on desmin and calpain, provided experimental basis for the influencing mechanism of protein oxidation on meat ageing.1.Influence of oxidation on the susceptibility of purified desmin to degradation by μ-calpain, caspase-3 and caspase-6This study was designed to investigate the effects of desmin oxidation on its degradation by proteolytic enzymes. Desmin was isolated from bovine muscle and exposed to varying oxidative conditions, and then incubated with either μ-calpain, caspase-3 or -6. The extent of protein degradation was subsequently determined using SDS-PAGE and western-blotting. Furthermore, the oxidative modification of the secondary structure of desmin was measured by circular dichroism (CD). Our results reveal that compared with the native desmin, degradation of oxidized desmin was enhanced by caspases, but suppressed by μ-calpain. The CD spectra of desmin showed that the content of a-helix decreased from 76.59% to 52.12% while random coil increased from 7.84% to 22.55% after oxidation. These findings demonstrate that oxidative modifications of desmin changed their susceptibility to μ-calpain, caspase-3 and -6 as well as their secondary structure.2. Effect of oxidation on the activity and autolysis of calpain during beef postmortem ageingThe aim of this experiment was to investigate the influence of oxidation on activity and autolysis of calpain during postmortem ageing. The Longissimus dorsi was excised from the carcass immediately after exsanguination. Then oxidizing agent with different concentrations (0 μmol/L H2O2; 50 mmol/L H2O2; 200 mmol/L H2O2; 500 mmol/L H2O2) was injected. The samples were vacuum packaged, stored at 4℃ for 0、1、3、7、14d, respectively. Then Casein Zymography and Western-Blotting were used to analyze the samples. The results showed that μ-calpain activity decreased and autolysis enhanced while m-calpain activity has undergone minor change as the oxidative level increased.. Therefore, oxidation inhibited the activity of μ-calpain and increased its autolysis during beef postmortem ageing.