Study On Extraction And Properties Of Superoxide Dismutase From Goose Blood

The most goose blood is producted by China in the world, so it has been rich in goose blood resources. Goose blood has high nutritional value, but a few has been developed goose blood tofu or goose blood meal et al, most were abandoned in the process. In this paper, goose blood was used for raw material, added appropriate protective agent and soluble film former, used mixer to stir and obtained hemolysis, after ethanol- chloroform and heat denature, getted superoxide dismutase (SOD) crude enzyme solution from goose blood, and then after acetone preliminary purification and DEAE-52 further purification, getted pure SOD. Last, I studied some properties of SOD from goose blood. Contents and results of research are as follows:First, the extraction of SOD from anticoagulant goose blood and coagulation goose blood were studied .Concluded that optimum conditions of extraction of SOD from anticoagulant goose blood as follow: heat treatment time 15min, heat treatment temperature 60℃, and the volume fraction of CuCl2 2.0%, SOD specific activity is 915.585U/mg.Optimum conditions of extraction of SOD from goose blood coagulation as follow: heat treatment time 15min, heat treatment temperature 60℃, and the volume fraction of CuCl2 2.0%, SOD specific activity is 1144.896U/mg. Therefore, specific activity of SOD from coagulation goose blood is higher(activity and purity is better). Furthermore, extracting SOD from coagulation goose blood did not need to add anticoagulants and separate blood cells, extraction process is relatively simple.Second, acetone was used to purificate SOD crude enzyme preliminary, and SOD specific activity was studied with different the volume ratio of acetone and SOD crude enzyme, Concluded that if the volume ratio of acetone and SOD crude enzyme is 1.0, SOD specific activity would be 1547.42 U/mg ,which is the highest. Then through Sephadex G-100 or DEAE-52 cellulose purified SOD, definited the most appropriate sample volume(2.5mL,5.0mL,7.5mL) and flow (1.0mL/min,1.5mL/min,2.0mL/min),The results show: when sample volume was 5.0mL and flow was 1.5mL/min, the purity of SOD was the better, SOD specific activity was 9257.67 U/mg, purification multiple was 95.7; after DEAE-52 cellulose purified SOD crude enzyme, SOD specific activity was 9783.362 U/mg and purification multiple was 101.1, therefore, the two purification methods were well, purification multiple has greatly improved, but DEAE-52 cellulose purification is the better.Third, SDS-PAGE electrophoresis and SOD activity staining were used to identify SOD purity and determine subunit molecular weight, the results showed that SOD has reached electrophoresis purity and subunit molecular weight is 16KD. Then I studied some physical and chemical properties of SOD, including determining the maximum UV absorption peak, sensitivity test of KCN and H₂O₂, SOD antioxidant activity and the impact of some factors, such as temperature,pH,metal ions. The results showed that maximum UV absorption peak of geese blood SOD is 258nm, it is sensitive to both KCN and H₂O₂, so goose blood SOD is belong to Cu/Zn-SOD; and SOD antioxidant activity is well, adding 50μL SOD can reach 50% inhibition rate for pyrogallol autoxidation rate. thermal stability of SOD is well, optimum temperature is 60℃; pH stability is also well, when pH is 6.0 to 9.0, the relatively enzyme activity of SOD is very high, but pH is too high or too low to protect SOD activity; different metal ions have different influence on SOD activity, the order of activation is Cu²⁺>Zn²⁺>K⁺>Mg²⁺>Ca²⁺>Al³⁺ , the order of inhibition is Sn²⁺>Pb²⁺>Fe³⁺>Na⁺.