Study On Purification And Chemical Modification Of SOD From Sheep Blood

The Superoxide dismutase (Superoxide dismutase, SOD) can catalyze the superoxide free radical (O2.-) to produce hydrogen peroxide(H₂O₂) and oxygen. H₂O₂ can be decomposed into H2O and O2 by catalase. The SOD preparations are widely utilized in the medical service, food and the cosmetics field. SOD's stability can be strengthened by the method of chemical modification in order to enlarge its application range. Inner Mongolia is rich in livestock resources with the amount of slaughtered sheep about 150 million each year. a large number of sheep blood resources are not fully utilized. Fresh sheep blood was used as the experimental materials for its abundant SOD. Firstly crude SOD products were obtained by using organic solvents to denature the other proteins and remove them. At the same time the key extraction conditions(optimum denaturing temperature and copper concentration) were explored. The crude SOD products were purified by column chromatography to get pure SOD .SOD is identified by the method of NBT-staining protein ,and the activity of SOD is determined by the method of pyrogallol. At last, sensitivity of the SOD to H202 and spectral scanning were analyzed., PEG-6000, bovine serum albumin and lauric acid were selected for the SOD modification. Three kinds of modified SOD were acquired. The amino acid composition analysis , modification rate, thermal stability and enzymolysis resistance of modified SOD were studied. The research results are as follows:1,1257mg SOD powder was obtained from 1000ml sheep blood. The total activity and specific activity are 79453U and 3871.4U/mg respectively. Investigation of physiochemical of the SOD showed that the ultraviolet absorption peak of the enzyme was found to be at 264nm and inhibited by H₂O₂. The results of non-denaturing gel electrophoresis and denaturing PAGE showed that two bands were SOD, and molecular weight of SOD subunits were 16.71KDa and15.97KDa respectively.2,It proved that SOD products,which were modificated by lauric acid, bovine serum albumin and PEG-6000 respectively, were still active ,and the molecular weight has changed by the determination of activity and SDS-PAGE. The modification rate of three kinds of modified SOD were 84.2%(lauric acid), 44.7%(bovine serum albumin)and 71.8%(PEG-6000 )respectively. The residual activity of them were 2943.7U/ml (lauric acid), 2791.5 U/ml(bovine serum albumin) and 2379.4 U/ml (PEG-6000) respectively. The residual activity(%) of SOD modified by lauric acid , bovine serum albumin and PEG-6000 were 89.6%, 84.8 % and 72.4% respectively, . The thermal stability of three kinds of SOD products,which were modified by lauric acid, bovine serum albumin and PEG-6000 respectively, decreased systematically.3,The possible modification sites of amino residues were predicted by using analyzing amino acid composition of SOD from sheep and three kind of modified SOD.The results can provide the theoretical basis to the chemical modification for SOD and the further utilization of animal blood resource.