猪血铜锌超氧化物歧化酶的纯化及其性质研究
|
摘要
本文采用有机溶剂(乙醇-氯仿)的方法进行猪血SOD粗酶液的制备，然后分别用两种不同的方法对粗酶液进行过柱纯化：第一种方法是用葡聚糖G100，G50和阴离子交换树脂依次先后进行SOD纯化，所用猪血60ml，可得SOD纯品约2.44mg，比活力达4420.7U／mg左右，纯化倍数约28.6倍左右，总活力回收率约48％；第二种方法是只用葡聚糖G100-G50进行串连柱层析，所用猪血5000ml，可得SOD纯品约275.6mg，比活力达4700U／mg左右，纯化倍数约31倍左右，总活力回收率约70％。纯化后的两种样品经聚丙烯酰胺凝胶电泳鉴定和酶活性测定，结果表明：两种方法分离纯化的SOD纯度高，活力也高，说明后者在前者的基础上发展起来后，更趋于合理。省去了阴离子交换不说，还极大的提高了总产量和活力得率，而且由三次操作简化成一次过柱即可，节省了药品及缓冲液用量，降低了劳动强度和电力消耗，减少了操作过程中的人为浪费，缩短了生产周期，与生产极为有利。因而不管从第一次的纯化来看，还是从文献上报道的来看，本实验的串联柱层析是较为合理有效的一种生产方法，值得推广。
用不同的温度与不同的时间对猪血SOD粗酶液进行处理，结果表明：猪血粗酶液的处理以变性温度55-65℃，变性时间为15～26分钟最好，此时杂蛋白去除量较大而酶的损失却相对较小，比活力较高。
本文采用聚丙烯酰胺凝胶电泳，巯基已醇-SDS-聚丙烯酰胺凝胶电泳，等电聚焦电泳以及各种凝胶层析的方法对猪血铜／锌超氧化物歧化酶进行了研究。结果表明：猪血只含一种Cu，Zn-SOD类型，其分子量为32112，有两个相同的亚基构成，本文所测得的亚基约为16293，约为分子量的一半。在非变性聚丙烯酰胺凝胶电泳中，酶谱带数为最多时达4条，在等电聚焦电泳中，最多时酶谱带数亦达4条，此酶在电泳条件下出现的多谱带现象可能是由亚基的解聚和构象异构体两种原因造成的。由此排除了其是由不同基因编码的同工酶或在转录后加工及分离纯化过程中形成肽链长短不一和酶分子侧链基团差异等微不均一性所造成的可能性。
The isolation of crude Cu, Zn-SOD can be started with organic solvents (ethanol-chloroform) in the classical manner. Then we take two different methods:the first one is that chromatography is carried out on Sephadex G100, G50 at room temperature with PH7. 6, 0. 02M phosphate buffer, respectively, Following this, ion-exchange chromatography is carried at pH7. 6 with 0. 02M and 0. 4M phosphate buffer;the second is that the crude enzyme runs on Sephadex G100-G50 column under the same experimental conditions(only with Sephadex G100, G50).At last polyacrylamide gel electrophoresis and SOD assay tell us both of products are highly purified SOD. These SOD activities are very high, too.
Crude hog superoxide dismutase treated with different temperatures and for different time, the result.shows as following: The treatment is most appropriate at 55-65 C and for 15-26 minutes for thermal denatured extracting SOD from hog blood and specific activi is very high.
In this thesis, we have done some research on the characterization of Cu, Zn-SOD from hog blood by polyacrylamide gel electrophoresis, electric focusing electrophoresis and dealing with some different reagents. The result shows as follows: one is that hog Cu, Zn-SOD has no isozymes and it contains the same two subunits , with molecular masses of 32112 daltons . the other is that the polymorphism of the Cu, Zn-SOD from hog blood is caused by polymerization and conformational isomers. So it has been found that this polymorphism of SOD was changed under different experimental conditions, which showed that the polymorphism was not caused by the difference of genes or the difference of length or side groups of the peptide chains which was caused during the processing after transcript or the purification. According to the
references about the protein research, we estimated that the possible reasons of the polymorphism were polymerization, conformational isomers and charge isomers. There was some research on the SOD charge isomers, but not on this polymorphism.
用不同的温度与不同的时间对猪血SOD粗酶液进行处理，结果表明：猪血粗酶液的处理以变性温度55-65℃，变性时间为15～26分钟最好，此时杂蛋白去除量较大而酶的损失却相对较小，比活力较高。
本文采用聚丙烯酰胺凝胶电泳，巯基已醇-SDS-聚丙烯酰胺凝胶电泳，等电聚焦电泳以及各种凝胶层析的方法对猪血铜／锌超氧化物歧化酶进行了研究。结果表明：猪血只含一种Cu，Zn-SOD类型，其分子量为32112，有两个相同的亚基构成，本文所测得的亚基约为16293，约为分子量的一半。在非变性聚丙烯酰胺凝胶电泳中，酶谱带数为最多时达4条，在等电聚焦电泳中，最多时酶谱带数亦达4条，此酶在电泳条件下出现的多谱带现象可能是由亚基的解聚和构象异构体两种原因造成的。由此排除了其是由不同基因编码的同工酶或在转录后加工及分离纯化过程中形成肽链长短不一和酶分子侧链基团差异等微不均一性所造成的可能性。
The isolation of crude Cu, Zn-SOD can be started with organic solvents (ethanol-chloroform) in the classical manner. Then we take two different methods:the first one is that chromatography is carried out on Sephadex G100, G50 at room temperature with PH7. 6, 0. 02M phosphate buffer, respectively, Following this, ion-exchange chromatography is carried at pH7. 6 with 0. 02M and 0. 4M phosphate buffer;the second is that the crude enzyme runs on Sephadex G100-G50 column under the same experimental conditions(only with Sephadex G100, G50).At last polyacrylamide gel electrophoresis and SOD assay tell us both of products are highly purified SOD. These SOD activities are very high, too.
Crude hog superoxide dismutase treated with different temperatures and for different time, the result.shows as following: The treatment is most appropriate at 55-65 C and for 15-26 minutes for thermal denatured extracting SOD from hog blood and specific activi is very high.
In this thesis, we have done some research on the characterization of Cu, Zn-SOD from hog blood by polyacrylamide gel electrophoresis, electric focusing electrophoresis and dealing with some different reagents. The result shows as follows: one is that hog Cu, Zn-SOD has no isozymes and it contains the same two subunits , with molecular masses of 32112 daltons . the other is that the polymorphism of the Cu, Zn-SOD from hog blood is caused by polymerization and conformational isomers. So it has been found that this polymorphism of SOD was changed under different experimental conditions, which showed that the polymorphism was not caused by the difference of genes or the difference of length or side groups of the peptide chains which was caused during the processing after transcript or the purification. According to the
references about the protein research, we estimated that the possible reasons of the polymorphism were polymerization, conformational isomers and charge isomers. There was some research on the SOD charge isomers, but not on this polymorphism.
引文
1. J.M. Mc Cord and .Fridovich, Superoxide diamutase , J, Boil chem, 1969,224:6049-6055.
2. McCord, J. M. and Fridovich, Ⅰ. Superoride dismutase: an enzymic function for erythrocuprein (hemocuprein) J. Biol. Chem, 1969, 193: 353-358
3．李文杰，超氧化物歧化酶在治疗超氧阴离子自由基所引起的疾病及抗衰老上的应用，中国药学杂志，1989，24(7)：397-400。
4. Michelson A M, et al. In Superoxide and Superoxide dismutase, ed by Michelson et al. London:Academic Press, 1977,467.
5. French, S.&Wilson, K.S..On treatment of negative intensity observations. Acta Crystallog. sect. A, 1978,34:517-525.
6．袁勤生、翁清清、王志友，猪血超氧化物歧化酶的纯化及其性质研究，生物化学与生物物理学报，1987，19(1)：22—26
7．李卫平、徐长法、王楠等，北京鸭红细胞铜锌超氧化物歧化酶电荷异构体的分离和某些性质，生物化学杂志，1995，11(3)：276-280
8．皱国林、钟晓凌、王东梅等，猪红细胞的铜锌超氧化物歧化酶纯化和性质的研究，武汉大学学报，1988，34(1)：115-120
9．陈来同，唐运，41种生物化学产品生产技术．北京：金盾出版社，1994：
10 袁勤生，超氧化的物歧化酶的来源及制备工艺中国药学杂志，1989，24(7)：395．
11．张大志，羊红细胞超氧化物歧化酶的研究，生物化学杂志，1991，7(2)：211—215
12．吕星，狗肝铜锌超氧化物歧化酶的纯化，生物化学杂志，1991，7(4)：496—500
13．施茵，超氧化物歧化酶的分离提取，生物技术，2002，12(1)：21—22．
14．吕星、陈吉中等，铜离子整合亲和层析纯化人铜锌超氧化物歧化酶，生物化学与生物物理进展，1994，21(3)：259—262
15. Konnert, J.H. A restrained-parameter structure-factor least-squares refinement procedure for large asymmetric units. Acta Crystallog. sect. A, 1976,32:614-617.
16. Lamzin, V.S.,Wilson, K.S., Automated refinement of protein models. Acta Crystallog. Sect. D, 1933, 49:129-147
17．张宏谭、竹钧，牛血超氧化物歧化酶生产工艺研究，内蒙古大学学报，2002，33(5)：567—571
18．胡滨、赵艳丽等，大肠杆菌发酵生产超氧化物歧化酶最佳条件研究，生物技术，2002，12(3)：33—35
19. Puget, K.,Michelson, A.M., Isolation of a new copper-containing superoxide dismutase, bacteriocuprein. Biochem. Biophys. Res. Commun. 1974,58:830-838.
20. Steinman, H. M. Cooper-zinc superoxide dismutase from Caulobacter crescentis-:A novel bacteriocuprein, form of the enzyme. J. Biol. Chem, 1982,257:10283-10293.
21．邓碧玉、袁勤生、李文杰，改良的连苯三酚自氧化测定超氧化物歧化酶活性的方法，生物化学与生物物理进展，1991，18(2)：163
22. Beck, B.L.,Tabatabai, L.B.,et al, A protein isolated from Brucella abortus is a copper zinc superoxide dismutase Biochemistry ;1990,29:372-376
23. Kroll, J.S.;Langford, P.R;LOYNDS, B.M. Copper-zinc superoxide dismutase of Haemophilus infhenzae and Haemophilus parainfluenzae. J. Bacteriol.. 1991,173:7449-7457.
24. Langford, P.R.;Loynds, B.M.; Kroll, J.S. Copper-zinc superoxide dismutase in Haemophihs species. J. Gen. Microbiol. 1992,183:517-522..
25. Steinman, H.M. Bacteriocuprein superoxide dismutase in pseudomonads. J. Bacteriol. 1985,162:1255-1260.
26. Steinman, H.M. Bacteriocuprein superoxide dismutase in pseudomonads.
J. Bacteriol. 1985,162:1255-1260.
27. Steinman, H.M.,Ely, B. Copper-zinc superoxide dismutase of Caulobacter crescentis:Cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme. J. Bacteriol.,1990,172:2901-2910.
28. Stabel, T.J., Sha, z.,et al, Periplasmic location of Bruscella abortus copper-zinc superoxide dismutase. Vet. Microbiol. 1994,38:307-314.
29. Benov, L.T., Friovich, I. Escherichia coli expresses a copper, zinc superoxide dismutase. J. Biol. Chem. 1994,269:25310-25314.
30. Benov, L.T. Chang, L.Y.,et al, Copper, zinc superoxide dismutase in Escherichia coli:Periplasmic localization. Arch. Biochem. Biophys. 1995,319:508-511.
31. Imlay, J.A. Linn, S. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J. Bacteriol. 1987,169:2967-2976.
32. Lowry, O.H.;Rosebrough, N.F.;et al, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951,193:265-275.
33．王爱国，大豆种子超氧化物歧化酶的研究[J]，植物生理学报，1983，(9)：77～84。
34．罗广华、王爱国，植物SOD的凝胶电泳及活性的显示，植物生理学通讯，1983(6)：44—45
35．杨唐斌、梅尚筠，超氧化物歧化酶活性正、负染色法的比较，生物化学与生物物理进展，1991，18(6)：468—470
36．陈汤杨、刘望夷，从超氧化物歧化酶的分布和结构看其分子进化，生物化学与生物物理进展，1996，23(5)：408—413
37．陶明煊、吴国荣、魏锦城，蓝、绿藻SOD同工酶类型及其进化，南京师大学报(自然科学版)，1999，22(4)：93
38..Desideri, A,.Felconi,M.,et al, Evolutionary conservativeness of electrostatic field in the Cu, Zn superoxide dismutase active site
J. Mol. Biol, 1992,223:332-352.
39. Stellwgen, E. and Wilgue, H, Biochemistry of Thermophily, (Friedman,S. M. ed.), Academic press, New York, 1978.
40. Konnert, J.H.,Hendrickson. W.A.,A restrained-parameter thermal-factor refinement procedure, Acta Crystallog. sect. A. 1980,36: 344-350
41．唐双焱、皱国林、金建明，牦牛血超氧化物歧化酶电泳多谱带现象研究，动物学报，2000，46(4)：422—430
42. Asahara, H.,Wistort, P.M., et al, Purification and characterization of the Escherichia coli endonuclease Ⅲ from the coloned nth gene, Biochemistry, 1989,28:4444-4449.
43．骆训懿、王晶翼、谢邦铁等，重组人超氧化物歧化酶化学修饰的初步研究，生物化学与生物物理进展，1993，20(6)：452—456
44．张恒，人EC-超氧化物歧化酶的克隆及高效表达，华东理工大学学报，2002，28(1)：39—42．
45．李益新、方允中、刘智峰，铜锌超氧化物歧化酶的重组研究，生物化学与生物物理学报，1984，16(5)：472—479．
46. Azab. H.A., Banci. L.,et al, Redox chemistry of superoxide dismutants on functionally relevant issues, Inorg. Chem. 1992, 31: 4649-4655.
47. Banci. L.,Bertini. I.,et al, X-ray NMR and molecular dynamics studies on reduced bovine superoxide dismutase:implications for the mechanism, Biochem. Biophys. Res. Commun, 1994, 202:1088-1095
48. Klug, D.,Rabani, J.&Fridovich, I.A direct demonstration of the catalytic action of bovine erythrocyte superoxide dismutase through the use of pulse radiolysis. J. Biol. Chem. 1972,247:4839-4842.
49. McAdam, M.E.,Fielden, E.M., et al, The involvement of the bridging imidazole in the catalytic mechanism of the action of bovine superoxide dismutase, Biochem. J. 1977, 167:271-274.
50. Osman, R.,Basch, H., On the mechanism of superoxide dismutase: a theoretical study. J. Am. Chem. Soc. 1984,106: 5710-5714.
51. Valentine, J.S.,Pantoliano, M.W. Protein-metalion interactions in cuprozinc proteins ,In Metal Ions in Biological Systems. (Sigel, H. ed.),1981, VOL. 3 pp 291-358. Dekker, New York.
52. Bertini. I.,Luchinat. C. et al, Evidence of the breaking of the copper-imidazole bridge in copper/ cobalt-substituted superoxide dismutase upon reduction of the Cu(Ⅱ) centers, J. Am. Chem. Soc, 1985, 107:2178-2179
53. Fee, J.A &Bull, C. Steady-state kinetic studies of superoxide dismutases. J. Biol. Chem. 1986,261:13000-13005.
54. Fridovich, I. Superoxide dismutase. Annu. Rev. Biochem. 1975,44:147-159.
55. Rotilio, G.,Finazzi. et al, Metal sites of copper proteins. Ligands of copper in hemocuprein. Biochemistry, 1971,10:616-621.
56. Tainer, J.A.,Getzoff, E.O.,Beem ,K.M.,et al. Determination and analysis of 2 structure of copper zinc superoxide dismutase. J. Mol. Biol. 1982,160:181-217
57. Tainer. J.A, Getzoff. E.D, et al. Structure and mechanism of copper ,zinc superoxide dismutase .Nature. 1983,306:284-287.
58．梁贤振、李连之、胡绪英等，牛红细胞SOD活性中心的紫外光谱研究，中国生物化学与分子生物学报，1998，14(2)：175-180．
2. McCord, J. M. and Fridovich, Ⅰ. Superoride dismutase: an enzymic function for erythrocuprein (hemocuprein) J. Biol. Chem, 1969, 193: 353-358
3．李文杰，超氧化物歧化酶在治疗超氧阴离子自由基所引起的疾病及抗衰老上的应用，中国药学杂志，1989，24(7)：397-400。
4. Michelson A M, et al. In Superoxide and Superoxide dismutase, ed by Michelson et al. London:Academic Press, 1977,467.
5. French, S.&Wilson, K.S..On treatment of negative intensity observations. Acta Crystallog. sect. A, 1978,34:517-525.
6．袁勤生、翁清清、王志友，猪血超氧化物歧化酶的纯化及其性质研究，生物化学与生物物理学报，1987，19(1)：22—26
7．李卫平、徐长法、王楠等，北京鸭红细胞铜锌超氧化物歧化酶电荷异构体的分离和某些性质，生物化学杂志，1995，11(3)：276-280
8．皱国林、钟晓凌、王东梅等，猪红细胞的铜锌超氧化物歧化酶纯化和性质的研究，武汉大学学报，1988，34(1)：115-120
9．陈来同，唐运，41种生物化学产品生产技术．北京：金盾出版社，1994：
10 袁勤生，超氧化的物歧化酶的来源及制备工艺中国药学杂志，1989，24(7)：395．
11．张大志，羊红细胞超氧化物歧化酶的研究，生物化学杂志，1991，7(2)：211—215
12．吕星，狗肝铜锌超氧化物歧化酶的纯化，生物化学杂志，1991，7(4)：496—500
13．施茵，超氧化物歧化酶的分离提取，生物技术，2002，12(1)：21—22．
14．吕星、陈吉中等，铜离子整合亲和层析纯化人铜锌超氧化物歧化酶，生物化学与生物物理进展，1994，21(3)：259—262
15. Konnert, J.H. A restrained-parameter structure-factor least-squares refinement procedure for large asymmetric units. Acta Crystallog. sect. A, 1976,32:614-617.
16. Lamzin, V.S.,Wilson, K.S., Automated refinement of protein models. Acta Crystallog. Sect. D, 1933, 49:129-147
17．张宏谭、竹钧，牛血超氧化物歧化酶生产工艺研究，内蒙古大学学报，2002，33(5)：567—571
18．胡滨、赵艳丽等，大肠杆菌发酵生产超氧化物歧化酶最佳条件研究，生物技术，2002，12(3)：33—35
19. Puget, K.,Michelson, A.M., Isolation of a new copper-containing superoxide dismutase, bacteriocuprein. Biochem. Biophys. Res. Commun. 1974,58:830-838.
20. Steinman, H. M. Cooper-zinc superoxide dismutase from Caulobacter crescentis-:A novel bacteriocuprein, form of the enzyme. J. Biol. Chem, 1982,257:10283-10293.
21．邓碧玉、袁勤生、李文杰，改良的连苯三酚自氧化测定超氧化物歧化酶活性的方法，生物化学与生物物理进展，1991，18(2)：163
22. Beck, B.L.,Tabatabai, L.B.,et al, A protein isolated from Brucella abortus is a copper zinc superoxide dismutase Biochemistry ;1990,29:372-376
23. Kroll, J.S.;Langford, P.R;LOYNDS, B.M. Copper-zinc superoxide dismutase of Haemophilus infhenzae and Haemophilus parainfluenzae. J. Bacteriol.. 1991,173:7449-7457.
24. Langford, P.R.;Loynds, B.M.; Kroll, J.S. Copper-zinc superoxide dismutase in Haemophihs species. J. Gen. Microbiol. 1992,183:517-522..
25. Steinman, H.M. Bacteriocuprein superoxide dismutase in pseudomonads. J. Bacteriol. 1985,162:1255-1260.
26. Steinman, H.M. Bacteriocuprein superoxide dismutase in pseudomonads.
J. Bacteriol. 1985,162:1255-1260.
27. Steinman, H.M.,Ely, B. Copper-zinc superoxide dismutase of Caulobacter crescentis:Cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme. J. Bacteriol.,1990,172:2901-2910.
28. Stabel, T.J., Sha, z.,et al, Periplasmic location of Bruscella abortus copper-zinc superoxide dismutase. Vet. Microbiol. 1994,38:307-314.
29. Benov, L.T., Friovich, I. Escherichia coli expresses a copper, zinc superoxide dismutase. J. Biol. Chem. 1994,269:25310-25314.
30. Benov, L.T. Chang, L.Y.,et al, Copper, zinc superoxide dismutase in Escherichia coli:Periplasmic localization. Arch. Biochem. Biophys. 1995,319:508-511.
31. Imlay, J.A. Linn, S. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J. Bacteriol. 1987,169:2967-2976.
32. Lowry, O.H.;Rosebrough, N.F.;et al, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 1951,193:265-275.
33．王爱国，大豆种子超氧化物歧化酶的研究[J]，植物生理学报，1983，(9)：77～84。
34．罗广华、王爱国，植物SOD的凝胶电泳及活性的显示，植物生理学通讯，1983(6)：44—45
35．杨唐斌、梅尚筠，超氧化物歧化酶活性正、负染色法的比较，生物化学与生物物理进展，1991，18(6)：468—470
36．陈汤杨、刘望夷，从超氧化物歧化酶的分布和结构看其分子进化，生物化学与生物物理进展，1996，23(5)：408—413
37．陶明煊、吴国荣、魏锦城，蓝、绿藻SOD同工酶类型及其进化，南京师大学报(自然科学版)，1999，22(4)：93
38..Desideri, A,.Felconi,M.,et al, Evolutionary conservativeness of electrostatic field in the Cu, Zn superoxide dismutase active site
J. Mol. Biol, 1992,223:332-352.
39. Stellwgen, E. and Wilgue, H, Biochemistry of Thermophily, (Friedman,S. M. ed.), Academic press, New York, 1978.
40. Konnert, J.H.,Hendrickson. W.A.,A restrained-parameter thermal-factor refinement procedure, Acta Crystallog. sect. A. 1980,36: 344-350
41．唐双焱、皱国林、金建明，牦牛血超氧化物歧化酶电泳多谱带现象研究，动物学报，2000，46(4)：422—430
42. Asahara, H.,Wistort, P.M., et al, Purification and characterization of the Escherichia coli endonuclease Ⅲ from the coloned nth gene, Biochemistry, 1989,28:4444-4449.
43．骆训懿、王晶翼、谢邦铁等，重组人超氧化物歧化酶化学修饰的初步研究，生物化学与生物物理进展，1993，20(6)：452—456
44．张恒，人EC-超氧化物歧化酶的克隆及高效表达，华东理工大学学报，2002，28(1)：39—42．
45．李益新、方允中、刘智峰，铜锌超氧化物歧化酶的重组研究，生物化学与生物物理学报，1984，16(5)：472—479．
46. Azab. H.A., Banci. L.,et al, Redox chemistry of superoxide dismutants on functionally relevant issues, Inorg. Chem. 1992, 31: 4649-4655.
47. Banci. L.,Bertini. I.,et al, X-ray NMR and molecular dynamics studies on reduced bovine superoxide dismutase:implications for the mechanism, Biochem. Biophys. Res. Commun, 1994, 202:1088-1095
48. Klug, D.,Rabani, J.&Fridovich, I.A direct demonstration of the catalytic action of bovine erythrocyte superoxide dismutase through the use of pulse radiolysis. J. Biol. Chem. 1972,247:4839-4842.
49. McAdam, M.E.,Fielden, E.M., et al, The involvement of the bridging imidazole in the catalytic mechanism of the action of bovine superoxide dismutase, Biochem. J. 1977, 167:271-274.
50. Osman, R.,Basch, H., On the mechanism of superoxide dismutase: a theoretical study. J. Am. Chem. Soc. 1984,106: 5710-5714.
51. Valentine, J.S.,Pantoliano, M.W. Protein-metalion interactions in cuprozinc proteins ,In Metal Ions in Biological Systems. (Sigel, H. ed.),1981, VOL. 3 pp 291-358. Dekker, New York.
52. Bertini. I.,Luchinat. C. et al, Evidence of the breaking of the copper-imidazole bridge in copper/ cobalt-substituted superoxide dismutase upon reduction of the Cu(Ⅱ) centers, J. Am. Chem. Soc, 1985, 107:2178-2179
53. Fee, J.A &Bull, C. Steady-state kinetic studies of superoxide dismutases. J. Biol. Chem. 1986,261:13000-13005.
54. Fridovich, I. Superoxide dismutase. Annu. Rev. Biochem. 1975,44:147-159.
55. Rotilio, G.,Finazzi. et al, Metal sites of copper proteins. Ligands of copper in hemocuprein. Biochemistry, 1971,10:616-621.
56. Tainer, J.A.,Getzoff, E.O.,Beem ,K.M.,et al. Determination and analysis of 2 structure of copper zinc superoxide dismutase. J. Mol. Biol. 1982,160:181-217
57. Tainer. J.A, Getzoff. E.D, et al. Structure and mechanism of copper ,zinc superoxide dismutase .Nature. 1983,306:284-287.
58．梁贤振、李连之、胡绪英等，牛红细胞SOD活性中心的紫外光谱研究，中国生物化学与分子生物学报，1998，14(2)：175-180．