| Superoxide dismutate (SOD, EC 1.15.1.1), one kind of metalloenzyme, which has been found ubiquitously among oxygen-metabolizing organisms, is most widely studied and reported in all enzymes we know. According to the type of metal in the active site of the enzymes, SOD has been classified into four types: Cu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD. It can catalyse the dismutation of superoxide anion radical to molecular oxygen and hydrogen peroxide. This enzyme has been proposed to play an important role in protecting living cells from the cytotoxic effects of the superoxide anion radical and other reactive oxygen species formed from the superoxide anion radical, such as hydrogen peroxide, the hydroxyl radical and singlet oxygen. SOD was related to caducity, tumours, autoimmunity diseases and radioprotection, so it has fine foreground of development and application. This text summarizes the researches about SOD, such as the type, distribution, chemical structure, and chemical character, the measurement of activity, content and applications of SOD.A superoxide dismutate (SOD) was purified to homogeneity from fresh camellia pollen by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose (DEAE-52), Sephadex G-100 and Phenyl Sepharose?6 Fast Flow columns. The purified SOD had about 70602 units of enzymatic activity and the specific activity was 4034 units per mg protein. Then the SOD obtained by Phenyl Sepharose? 6 Fast Flow chromatography was dialyzed, freeze-dried. The purified SOD showed only one protein band by SDS-PAGE and isoelectric focusing electrophoresis. This explained that purified SOD was homogeneous. Finally 27.9% of the SOD activity was recovered. SOD was 81.8-fold purified.Protein concentration was determined by the Folin method using bovine serum albumin as the standard.SOD activity was assayed by the pyrogallol autoxidation method. One unit of SOD enzymatic activity was defined as the amount of enzyme inhibited the half of the rate of pyragallol autoxidation per min at 325 nm, 25℃.The protein was visualized by staining with Coomassie brilliant blue R 250 and whereas SOD activity was negatively stained by the riboflavin mediated photoreduction of Nitroblue Tetrazolium.Cu/Zn-SOD is sensitive to cyanide and hydrogen peroxide, while Fe -SOD is inhibited by hydrogen peroxide but not by cyanide. Mn-SOD, on the other hand, is inhibited by chloroform-alcohol but not by either cyanide or hydrogen peroxide. 10mmol/L KCN restrained 99% activity of purified SOD from Camellia Pollen, and 5mmol/L H2O2 restrained completely the activity of purified enzyme. It appeared to be Cu/Zn-SOD according to its specific sensitivitives to hydrogen peroxide and cyanogens potassium.Molecular weight was determined by gel filtration on a Sephadex G-100 column (1.0×100 cm). The column was equilibrated with 25 mM potassium phosphate, pH 7.8, and calibrated with the following molecular weight standards: ribonuclease A (13,700 Da), chymotrypsinogen A (25,000 Da), ovalbumin (43,000 Da), bovine serum albumin (67,000 Da), and Blue Dextran 2000. The molecular weight of purified SOD was found to be 70.0kD by the method. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight and subunit weight of the SOD, which was purified to homogeneity from camellia pollen, were 69.5kD and 34.7kD respectively. It was a conclusion, which was composed of two subunits with the same molecular weights.Isoeletric point of the purified SOD was pH 4.1 by isoelectrofocusing electrophoresis.Molecular stability experiments searched the effects of pH, temperature, chemical reagents, metal ions and ion intensity to SOD. The results showed that the stability of our SOD is better than other enzymes. The optimum temperature and pH were 25℃and 6.6 respectively. The activity of the enzyme lost entirely when pH was higher than 11. The enzyme showed any activity in 5% EDTA, so the mental ions must play an important role in the active site. The subunit was depolymerizated in 0.5% P-mercaptoethanol, and the activity of the enzyme was highly effected by P-mercaptoethanol but not by 1%SDS, 4 mol/L urea and ion intensity. Therefore it was demonstrated that the purified SOD was Cu/Zn-SOD.10 mg superoxide dismutase was hydrolyzed for 24 h at 110℃with 5.7mol/L HC1 and then determined by amino acid automatic analyzer. Comparing with the amino acid compositions reported for other superoxide dismutases, the enzyme exhibits notable differences, and the SOD did contain slightly higher values of Tryptophan,Phenylalanine,Tyrosine.The N-terminal amino acid of the enzyme was identified to be Gly by DNS-Cl method.Optical absorption spectra was obtained with a 760 CRT recording spectrophotometer. The purified SOD has the characteristic absorption spectrum of Cu/Zn-SOD. Namely, the maximum absorbance apex of the purified SOD was at 274 nm in extraviolet light.Circular dichroism (CD) spectra were obtained with a JASCO J 810 recording spectropolarimeter. CD spectrum was investigated and found thatα-Helix,β-sheet,β-turn and Random 21.8%, 32.2%, 12.7% and 33.3% respectively.The ultraviolet spectra of the superoxide dismutate from camellia pollen ultraviolet spectra 760CRT were determined. The effects of different modification reagents on its catalytic activity were studied.Diethyl pyrocarbonate (DEPC) inactivated the SOD. The inactivation kinetics of the enzyme by DEPC was pseudo-first order one with respect to incubation time. Activity of modified the SOD could be restored by treatment with hydroxylamine. The ultraviolet difference spectrum of modified versus native enzyme revealed the formation of ethoxyformal histidine residues during inactivation. Kinetic data indicates that only one molecule of DEPC is involved in binding to each active unit of enzyme during inactivation. The result indicated that two histidine residue may be essential for supporting catalytic function of the SOD.Treatment of superoxide dismutate from camellia pollen with 1, 2-cyclohexanodine (CHD) could markedly decrease its activity. This inactivation depended on the presence of borate (0.1 mol/L) and the pH at which the modification reaction proceeded. The inactivation rate followed the pseudo first order kinetics with respect to reaction time and modifier concentration. It was demonstrated by the method of Keech and Farrant that only one molecule of 1,2-cyclohexanodine reacting with each active unit to produce an inactivated enzyme-modifier complex. The chemical modification kinetic data indicated that two Arginine groups were related to the enzyme activity.The inactivation was reversed when 1,2-cyclohexanodine and borate were removed by dialysis.Purified superoxide dismutate from camellia pollen was inactivated by amino group reagents, 2, 4, 6-trinitrobenzene sulfonate (TNBS). Observation of the absorption spectrum of the modified enzyme showed that it was the lysine residue.Chloramine-T (Ch-T) specific modifiers of methionine residue inhibited the activity of the SOD. The enzyme inhibited by Ch-T could be reactivated by 0.35 mol/L hydroxylamine (30 min). From these results we conclude that the inhibition of SOD by Ch-T is due to the modification of the methionine residue in the active center of the enzyme, and that methionine is essential to the activity of the SOD.The chemical modification of superoxide dismutate was studied with PCMB,IAA and IAM as the modification reagents on its SH group. The results indicate that SH group is not the activity dependent group. The inactivated enzyme could be treatment with IAA (10mmol/L) for 2 h, which meant that histidyl residues was modified by IAA but not SH group residues.Purified superoxide dismutate from camellia pollen was inactivated by water-soluble 1-ethy-3- (3-dimethylaminoprgpyl) carbodiimide (EDC). The inactivation kinetics of the enzyme by EDC was first order with respect to incubation time and inhibitor concentration. Kinetic date indicated that only two molecules of EDC were involved in binding to the enzyme during the inactivation process. The chemical modification kinetic data indicated that four carboxyl groups were related to the enzyme activity.Tryptophan residues in the SOD were modifid by N-bromosuccinimide (NBS). The results indicate that there are 21 Trp residues in SOD, seven of which are located on the surface of the enzyme. Glycerine could protect the combination of SOD and NBS. Fluorescence intensity of SOD receded continuously with adding NBS, but the figure of emission spectrum and the largest emission wavelength did not vary obviously.The superoxide dismutate from camellia pollen was selectively modified by 1, 4-Dithiothreitol (DTT) and N-acetylimidazole (N-AI), and changes in the activities of the enzyme have been detected. The DTT was found without any inhibition effect. The N-AI was found to have little inhibition effect. All of these suggested that tyrosine residue wasn't indispensable functional groups of SOD, and partial disulfide bonds were essential for the function of the enzyme.In conclusion, we purified a SOD from camellia pollen to homogeneity, identified its subtype and characterized its structure for the first time. These results have provided therotical basis for the mechanism of pollen's function in anti-senium at the molecular level and have enlarged the study of SOD.
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