金属蛋白制备及性质研究
|
摘要
金属蛋白—含有金属的蛋白—生命过程都离不开金属蛋白，人体内的蛋白大约1/3是金属蛋白，它对生命活动的基本过程很重要，包括DNA合成、新陈代谢、消除毒素以及生命所需的碳、氧和氮的化学转化。许多疾病是由于金属失衡或关键金属酶失活引起的，金属蛋白的制备和性能研究是理解其生理功能的前提。
针对转铁蛋白提取，超氧化物歧化酶（superoxide dismutase，SOD, EC1.15.1.1）的提取及性能和超氧化物歧化酶模拟酶的合成进行了研究，转铁蛋白是铁结合的血清糖蛋白，调控体液中游离铁水平，超氧化物歧化酶能将有害的超氧阴离子自由基歧化为无害的分子氧和双氧水。转铁蛋白和超氧化物歧化酶与越来越多的疾病相关联，包括炎症、癌症和糖尿病，因此它们在医药、营养和化妆行业有重要作用。转铁蛋白和超氧化物歧化酶从粗品通过两步纯化可获取纯品，第一步，热激法、硫酸铵沉淀法用于粗酶液的初步纯化。进一步纯化包括凝胶过滤层析、离子交换层析和亲和层析。得到的主要结论如下：
（1）鸭血清转铁蛋白经过两步纯化，热激和固定化金属螯合亲和层析，能获得光谱纯的鸭血清转铁蛋白。
（2）南瓜瓤是制备超氧化物歧化酶的新的、廉价的和可再生的资源。测试药食两用蔬菜中超氧化物歧化酶的活性，发现有些蔬菜，如南瓜瓤、南瓜果肉、南瓜籽和大蒜SOD每克样品（干基）超氧化物歧化酶酶活都高于500IU，属于富含超氧化物歧化酶的药食两用植物。其中南瓜瓤和南瓜果肉都高于南瓜籽；南瓜瓤和南瓜果肉都高于大蒜；而南瓜瓤中超氧化物歧化酶酶活最高。南瓜瓤是生产南瓜粉、南瓜籽和南瓜籽油等的副产品，是提取超氧化物歧化酶的廉价资源。
（3）建立了从南瓜瓤提取超氧化物歧化酶的简单而有成本效益的纯化方法。南瓜瓤用磷酸缓冲液浸提，经热激与硫酸铵沉淀耦合和亲和层析，能获得电泳纯的超氧化物歧化酶纯品，纯化倍数达到87，酶活收率为42%，比活为2884IU·mg-1蛋白。
（4）南瓜瓤超氧化物歧化酶是首次发现源于植物的糖基化SOD。南瓜瓤超氧化物歧化酶是同型二聚体，由两条相同的亚基组成，用MicrOTOF-Q质谱仪测得亚基分子量为17477Da，用凝胶过滤层析测得超氧化物歧化酶分子量为35.0kDa。南瓜瓤超氧化物歧化酶是Cu,Zn-SOD，每条亚基含有约1个铜和1个锌，并且对H2O2敏感。南瓜瓤Cu,Zn-SOD是糖蛋白，其分子量较其他植物Cu,Zn-SOD的高，pH稳定性范围较其他植物Cu,Zn-SOD的宽。在25℃，2h，南瓜瓤Cu,Zn-SOD在pH4～12保持其活性，在pH7.8，45min，南瓜瓤Cu,Zn-SOD在低于50℃保持其活性。南瓜瓤Cu,Zn-SOD活性被SDS和NaN3轻微抑制，而Na3BO3和柠檬酸钠对其活性没有影响。由此，被当作废弃物的南瓜瓤，是提取Cu,Zn-SOD的优质原料，可被化妆、医药和营养行业商业开发。
（5）合成了槲皮素铜及槲皮素锌配合物，然而，由于其低活性，它们不能作为超氧化物歧化酶模拟化合物，也许可以作为电荷调节剂。鸭血清转铁蛋白和南瓜瓤Cu,Zn-SOD被成功纯化，它们是金属结合的糖蛋白。
Metalloproteins—proteins containing metal atoms—are involved in a wide range ofimportant biological processes. Approximately one-third of the proteins in the human bodycontain metals. These metalloproteins are essential for the basic processes of life, includingDNA synthesis, metabolism, detoxification, and the chemical transformations of nitrogen,oxygen, and carbon molecules required for life. Many diseases are due to metal imbalances orinactivity of critical metalloenzymes. An understanding of physiologyical functions is achievedthrough production and characterization of metalloproteins.
Our focus is on isolation of transferrin, isolation and characterization of superoxidedismutase (superoxide dismutase, SOD, EC1.15.1.1) and synthesis of biomimetic complexes ofSOD. Transferrin is iron-binding blood plasma glycoprotein that controls the level of free ironin biological fluids. SOD can catalyze the dismutation of the harmful superoxide anion intomolecular oxygen and hydrogen peroxide. Transferrin and SOD have been associated with agrowing number of diseases such as inflammation, cancer, diabete, so they play an importantrole in pharmaceutical, nutraceutical and cosmeceutical industries. Transferrin and SOD fromcrude extract were purified in two steps. The first purification step of the crude extract wasachieved by heat treatment, ammonium sulfate precipitation. Further purification was achievedwith gel filtration chromatography, ion exchange chromatography or affinitychromatography. The main results are as follows:
(1) Transferrin from duck serum was purified in two steps. The process included the heattreatment and immobilized copper ion affinity membrane chromatography, yielding thespectrographically pure duck serum transferrin.
(2) Pumpkin stringy pulp was a new and cheap biorenewable resource of production ofSOD. SOD activity of different vegetables which used as both food and medicine was examed.The results showed that some vegetables, such as pumpkin flesh, pumpkin seed, pumpkinstringy pulp and garlic, were found more SOD activity of500IU·g-1(dry matter basis). SODactivity of pumpkin flesh and pumpkin stringy pulp was much more than that of anotherSOD-abundant plant, garlic. SOD activity of pumpkin stringy pulp was much more than that ofpumpkin flesh and pumpkin seed. Pumpkin stringy pulp is a by-product of those manufactorieswhich produce pumpkin flour, pumpkin seed, pumpkin seed oil, and so on. Therefore, pumpkinstringy pulp could be cheap and viable source of enzyme production.
(3) A simple and cost-effective way to purify SOD from pumpkin stringy pulp wasproposed. The stepwise isolation and purification procedure consists of phosphate buffer extract,heat treatment along with ammonium sulfate precipitation and immobilized metal affinitychromatography, yielding a87-fold purification (activity recovery of42%) and a single band upon SDS-PAGE and native-PAGE. The enzyme had2884U mg1protein-specific activity.
(4) The pumpkin pulp SOD is the first identified naturally glycosylated enzyme from plant.The enzyme had a molecular mass of35.0kDa, as determined by gel filtration chromatography.It was a dimeric enzyme with two identical subunits of17477Da, as measured usingMicrOTOF-Q mass spectrometer. The purified enzyme, which was characterized as aH2O2-sensitive SOD, contained approximately one copper atom and one zinc atom per subunit,indicating that it belongs to the Cu,Zn-SOD. Pumpkin pulp Cu,Zn-SOD is a glycosylatedenzyme, which possesses higher molecular mass and wider range of pH dependence of theactivity than other plant Cu,Zn-SOD. This naturally glycoprotein remained its activity at pH4–12at25°C for2h and the enzyme remained its activity lower than50°C at pH7.8for45min. The enzyme was slightly inhibited by SDS and NaN3, but was not inhibited by Na3BO3and sodium citrate. Therefore, these results suggested that pumpkin stringy pulp which isconsidered as a waste can be used as a rich source of Cu,Zn-SOD and can be commerciallyexploited in cosmeceuticals, pharmaceuticals and neutraceuticals.
(5) Quercetin-Cu complex and quercetin-Zu complex were synthesized. However, they arenot biomimetic complexes of SOD because of their low activity. Perhaps, they could be chargecontrol agents. Duck serum transferrin and pumpkin pulp Cu,Zn-SOD are separatedsuccessfully. They are metal-binding glycoproteins.
针对转铁蛋白提取，超氧化物歧化酶（superoxide dismutase，SOD, EC1.15.1.1）的提取及性能和超氧化物歧化酶模拟酶的合成进行了研究，转铁蛋白是铁结合的血清糖蛋白，调控体液中游离铁水平，超氧化物歧化酶能将有害的超氧阴离子自由基歧化为无害的分子氧和双氧水。转铁蛋白和超氧化物歧化酶与越来越多的疾病相关联，包括炎症、癌症和糖尿病，因此它们在医药、营养和化妆行业有重要作用。转铁蛋白和超氧化物歧化酶从粗品通过两步纯化可获取纯品，第一步，热激法、硫酸铵沉淀法用于粗酶液的初步纯化。进一步纯化包括凝胶过滤层析、离子交换层析和亲和层析。得到的主要结论如下：
（1）鸭血清转铁蛋白经过两步纯化，热激和固定化金属螯合亲和层析，能获得光谱纯的鸭血清转铁蛋白。
（2）南瓜瓤是制备超氧化物歧化酶的新的、廉价的和可再生的资源。测试药食两用蔬菜中超氧化物歧化酶的活性，发现有些蔬菜，如南瓜瓤、南瓜果肉、南瓜籽和大蒜SOD每克样品（干基）超氧化物歧化酶酶活都高于500IU，属于富含超氧化物歧化酶的药食两用植物。其中南瓜瓤和南瓜果肉都高于南瓜籽；南瓜瓤和南瓜果肉都高于大蒜；而南瓜瓤中超氧化物歧化酶酶活最高。南瓜瓤是生产南瓜粉、南瓜籽和南瓜籽油等的副产品，是提取超氧化物歧化酶的廉价资源。
（3）建立了从南瓜瓤提取超氧化物歧化酶的简单而有成本效益的纯化方法。南瓜瓤用磷酸缓冲液浸提，经热激与硫酸铵沉淀耦合和亲和层析，能获得电泳纯的超氧化物歧化酶纯品，纯化倍数达到87，酶活收率为42%，比活为2884IU·mg-1蛋白。
（4）南瓜瓤超氧化物歧化酶是首次发现源于植物的糖基化SOD。南瓜瓤超氧化物歧化酶是同型二聚体，由两条相同的亚基组成，用MicrOTOF-Q质谱仪测得亚基分子量为17477Da，用凝胶过滤层析测得超氧化物歧化酶分子量为35.0kDa。南瓜瓤超氧化物歧化酶是Cu,Zn-SOD，每条亚基含有约1个铜和1个锌，并且对H2O2敏感。南瓜瓤Cu,Zn-SOD是糖蛋白，其分子量较其他植物Cu,Zn-SOD的高，pH稳定性范围较其他植物Cu,Zn-SOD的宽。在25℃，2h，南瓜瓤Cu,Zn-SOD在pH4～12保持其活性，在pH7.8，45min，南瓜瓤Cu,Zn-SOD在低于50℃保持其活性。南瓜瓤Cu,Zn-SOD活性被SDS和NaN3轻微抑制，而Na3BO3和柠檬酸钠对其活性没有影响。由此，被当作废弃物的南瓜瓤，是提取Cu,Zn-SOD的优质原料，可被化妆、医药和营养行业商业开发。
（5）合成了槲皮素铜及槲皮素锌配合物，然而，由于其低活性，它们不能作为超氧化物歧化酶模拟化合物，也许可以作为电荷调节剂。鸭血清转铁蛋白和南瓜瓤Cu,Zn-SOD被成功纯化，它们是金属结合的糖蛋白。
Metalloproteins—proteins containing metal atoms—are involved in a wide range ofimportant biological processes. Approximately one-third of the proteins in the human bodycontain metals. These metalloproteins are essential for the basic processes of life, includingDNA synthesis, metabolism, detoxification, and the chemical transformations of nitrogen,oxygen, and carbon molecules required for life. Many diseases are due to metal imbalances orinactivity of critical metalloenzymes. An understanding of physiologyical functions is achievedthrough production and characterization of metalloproteins.
Our focus is on isolation of transferrin, isolation and characterization of superoxidedismutase (superoxide dismutase, SOD, EC1.15.1.1) and synthesis of biomimetic complexes ofSOD. Transferrin is iron-binding blood plasma glycoprotein that controls the level of free ironin biological fluids. SOD can catalyze the dismutation of the harmful superoxide anion intomolecular oxygen and hydrogen peroxide. Transferrin and SOD have been associated with agrowing number of diseases such as inflammation, cancer, diabete, so they play an importantrole in pharmaceutical, nutraceutical and cosmeceutical industries. Transferrin and SOD fromcrude extract were purified in two steps. The first purification step of the crude extract wasachieved by heat treatment, ammonium sulfate precipitation. Further purification was achievedwith gel filtration chromatography, ion exchange chromatography or affinitychromatography. The main results are as follows:
(1) Transferrin from duck serum was purified in two steps. The process included the heattreatment and immobilized copper ion affinity membrane chromatography, yielding thespectrographically pure duck serum transferrin.
(2) Pumpkin stringy pulp was a new and cheap biorenewable resource of production ofSOD. SOD activity of different vegetables which used as both food and medicine was examed.The results showed that some vegetables, such as pumpkin flesh, pumpkin seed, pumpkinstringy pulp and garlic, were found more SOD activity of500IU·g-1(dry matter basis). SODactivity of pumpkin flesh and pumpkin stringy pulp was much more than that of anotherSOD-abundant plant, garlic. SOD activity of pumpkin stringy pulp was much more than that ofpumpkin flesh and pumpkin seed. Pumpkin stringy pulp is a by-product of those manufactorieswhich produce pumpkin flour, pumpkin seed, pumpkin seed oil, and so on. Therefore, pumpkinstringy pulp could be cheap and viable source of enzyme production.
(3) A simple and cost-effective way to purify SOD from pumpkin stringy pulp wasproposed. The stepwise isolation and purification procedure consists of phosphate buffer extract,heat treatment along with ammonium sulfate precipitation and immobilized metal affinitychromatography, yielding a87-fold purification (activity recovery of42%) and a single band upon SDS-PAGE and native-PAGE. The enzyme had2884U mg1protein-specific activity.
(4) The pumpkin pulp SOD is the first identified naturally glycosylated enzyme from plant.The enzyme had a molecular mass of35.0kDa, as determined by gel filtration chromatography.It was a dimeric enzyme with two identical subunits of17477Da, as measured usingMicrOTOF-Q mass spectrometer. The purified enzyme, which was characterized as aH2O2-sensitive SOD, contained approximately one copper atom and one zinc atom per subunit,indicating that it belongs to the Cu,Zn-SOD. Pumpkin pulp Cu,Zn-SOD is a glycosylatedenzyme, which possesses higher molecular mass and wider range of pH dependence of theactivity than other plant Cu,Zn-SOD. This naturally glycoprotein remained its activity at pH4–12at25°C for2h and the enzyme remained its activity lower than50°C at pH7.8for45min. The enzyme was slightly inhibited by SDS and NaN3, but was not inhibited by Na3BO3and sodium citrate. Therefore, these results suggested that pumpkin stringy pulp which isconsidered as a waste can be used as a rich source of Cu,Zn-SOD and can be commerciallyexploited in cosmeceuticals, pharmaceuticals and neutraceuticals.
(5) Quercetin-Cu complex and quercetin-Zu complex were synthesized. However, they arenot biomimetic complexes of SOD because of their low activity. Perhaps, they could be chargecontrol agents. Duck serum transferrin and pumpkin pulp Cu,Zn-SOD are separatedsuccessfully. They are metal-binding glycoproteins.
引文
[1] Holmberg C G, Lauerll C B. Studies on the serum capacity of serum to bind iron[J]. ActaPhysiologica Scandinavica,1945,10:227-228.
[2] Gomme P T, McCann K B, Bertolini J. Transferrin: structure, function and potentialtherapeutic actions[J]. Drug Discovery Today,2005,10(4):267-273.
[3]王丛珠,王登平.转铁蛋白介导作用的应用进展[J].放射免疫学杂志,2003,16(5):304-306.
[4]章华础,肖祥熊.转铁蛋白放射免疫分析的临床应用[J].放射免疫学杂志,1997,10(6):370-373.
[5]廖继佩.乳铁蛋白Lf抗病毒感染研究进展[J].国外医学病毒学分册,2003,10(6):180-183.
[6]张宏伟,刘俊涛,王晓航.转铁蛋白和转铁蛋白受体的研究进展[J].国外医学临床生物化学与检验学手册,2000,21(4):183-184.
[7] Gupta R, Rastogi S, Nagar R, et al. Dietary and serum iron, body iron stores and coronaryheart disease [J]. The Journal of the Association of Physicians of India,2000,48(5):489-492.
[8] Harris W R, Messori L. A comparative study of aluminum(III), gallium(III), indium(III),and thallium(III) binding to human serum transferring[J]. Coordination Chemistry Reviews,2002,228(2):237-262.
[9] Messori L, Orioli P, Banholzer V, et al. Formation of titanium(IV) transferrin by reaction ofhuman serum apotransferrin with titanium complexes[J]. FEBS Letters,1999,442(2-3):157-161.
[10] Sadava D, Phillips T, Lin C, et al. Transferrin overcomes drug resistance to artemisinin inhuman small-cell lung carcinoma cells[J]. Cancer Letters,2002,179(2):151-156.
[11]冯佑民,孙嘉琳,童春香,等.转铁蛋白结构与功能的研究Ⅰ.猪转铁蛋白的分离纯化及在无血清细胞培养中的应用[J].生物化学与生物物理学报,1987,19(4):322-327.
[12] Ii I, Kimura I, Ozawa E. A myotrophic protein from chick embryo extract: Its purification,identity to transferrin, and indispensability for avian myogenesis[J]. DevelopmentalBiology,1982,94(2):366-377.
[13]王革新,孙南翔,王者鹏,等.人血浆转铁蛋白的分离与纯制[J].化学试剂,1991,13(4):244-247.
[14] McCann K B, Hughes B, Wu J, et al. Purification of transferrin from Cohn supernatant Iusing ion-exchange chromatography[J]. Biotechnology And Applied Biochemistry,2005,42(3):211-217.
[15] Chung M C-M, Chan S-L, Sfflmzu S. Purification of transferrins and lactoferrin usingDEAE Affi-Gel blue[J]. International Journal of Biochemistry,1987,23(5-6):609-616.
[16] Winzerling J J, Pham D Q-D, Kunz S, et al. Purification of Recombinant Insect Transferrinfrom Large Volumes of Cell Culture Medium Using High Capacity Ni2+-DipicolylamineGel[J]. Protein Expression and Purification,1996,7(2):137-142.
[17] Jain S, Gupta M N. An integrated process for separation of major and minor proteins fromgoat serum[J]. Applied Biochemistry and Biotechnology,2005,125(1):53-62.
[18] Ardehali R, Shi L, Janatova J, et al. The inhibitory activity of serum to prevent bacterialadhesion is mainly due to apo-transferrin[J]. Journal of Biomedical Materials ResearchPart A,2003,66(1):21-28.
[19] Lim H J, Kim Y K, Hwang D S, et al. Expression of functional human transferrin in stablytransfected Drosophila S2cells[J]. Biotechnology Progress,2004,20(4):1192-1197.
[20] McCord J M, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein(hemocuprein)[J]. Journal of Biological Chemistry,1969,244(22):6049-6055.
[21] Harman D. Aging: a theory based on free radical and radiation chemistry[J]. Journal ofGerontology,1956,11(3):298-300.
[22] Van Camp W, Inzé D, Van Montagu M. The regulation and function of tobacco superoxidedismutases [J]. Free Radical Biology Medicine,1997,23(3):515-520.
[23] Fester T, Schuster W. Potato mitochondrial manganese superoxide dismutase is anRNA-binding protein[J]. Biochemistry and Molecular Biology International,1995,36(1):67-75.
[24] Parker M W, Blake C C. Iron-and manganese-containing superoxide dismutases can bedistinguished by analysis of their primary structures[J]. FEBS Letters,1988,229(2):377-382.
[25]袁勤生.超氧化物歧化酶[M].上海：华东理工大学出版社，2009,170-192.
[26] Youn H D, Kim E J, Roe J H, et al. A novel nickel-containing superoxide dismutase fromstreptomyces spp[J]. Journal of Biochemistry,1996,318:889-896.
[27] Skarpanska-Stejnborn A, Pilaczynska-Szczesniak L, Basta P, et al. Effects of OralSupplementation With Plant Superoxide Dismutase Extract on Selected Redox Parametersand an Inflammatory Marker in a2,000-m Rowing-Ergometer Test. International Journalof Sport Nutrition and Exercise Metabolism,2011,124-134.
[28] Van Camp W, Capian K, Van Montagu M, et al. Enhancement of oxidative stress tolerancein transgenic tobacco plants over producing Fe superoxide dismutase in chlorop lasts [J].Plant Physiology,1996,112(4):1703-1714.
[29]赵晓瑜,宋小青,刘建荣.玉米Mn-SOD的分离纯化与性质分析[J].河北大学学报(自然科学版),2009,29(3):324-328.
[30]宫霞,孙淑斌,孔凡华.小白菜中超氧化物歧化酶的提取及稳定性[J].曲阜师范大学学报,1997,23(1):97-99.
[31] Wang J, Liu J, Lu J, et al. Isolation and purification of superoxide dismutase from garlicusing two-stage ultrafiltration[J]. Journal of Membrane Science,2010,352(1-2):231-238.
[32] He N, Li Q B, Sun D H, et al. Isolation, purification and characterization of superoxidedismutase from garlic[J]. Biochemical Engineering Journal,2008,38(1):33-38.
[33] Sheng L Q, Tong H W, Zheng X Y, et al. Purification and characterization of cytosolicisoenzyme III of Cu,Zn-superoxide dismutase from tobacco leaves[J]. Plant Science,2004,167(6):1235-1241.
[34]王中凤,肖厚荣,杨红,等.仙人掌SOD提取条件及提取过程活性稳定性研究[J].食品与发酵工业,2008,34(7):156-158.
[35] Haddad N I A, Yuan Q. Purification and some properties of Cu,Zn superoxide dismutasefrom Radix lethospermi seed, kind of Chinese traditional medicine[J]. Journal ofChromatography B,2005,818(2):123-131
[36] Vyas D, Kumar S. Purification and partial characterization of a low temperature responsiveMn-SOD from tea (Camellia sinensis (L.) O. Kuntze)[J]. Biochemical and BiophysicalResearch Communications,2005,329(3):831-838.
[37] Kumar S, Malhotra S P. Partial purification of superoxide dismutase and peroxidase fromber (Zizyphus mauritiana Lamk.) fruit using anion exchange chromatography[J].Physiology and Molecular Biology of Plants,2008,14(3):167-172.
[38] Kochhar S, Kochhar V. Identification and characterization of a super-stable Cu–Zn SODfrom leaves of turmeric (Curcuma longal)[J]. Planta,2008,228(2):307-318
[39] Babitha M.P, Prakash H.S, et al. Purification and partial characterization of manganesesuperoxide dismutase from downy mildew resistant pearl millet seedlings inoculated withSclerospora graminicola[J]. Plant Science,2002,163(4):917-924.
[40]毛绍春,李竹英,李聪.酸果蔓中SOD酶的提取及活性测定[J].食品与机械,2007,23(1):26-46.
[41]区炳庆,何丽烂,任吉君.南瓜种子超氧化物歧化酶某些性质的研究[J].广西植物,2004,24(5):463-465.
[42]卢运超,黄兆峰.南瓜粉冲剂的研制及临床应用[J].时珍国药研究,1997,8(3):264-265.
[43]吴素玲,孙晓明,金敬宏.南瓜品质资源的分析测试[J].中国野生植物资源,2002,21(2):53-54.
[44]王燕,车振明.南瓜的功能特性及其深加工[J].食品研究与开发,2005,26(3):7-10.
[45]卢颖,王永勤,任智捷.南瓜功能成分研究的进展及在医药领域中的应用[J].食品与药品,2005,7(7A):29-32.
[46]张建农,满艳萍.南瓜果实营养成分测定与分析[J].甘肃农大学学报1994,34(3):300-302.
[47]吴增茹,金同铭.用高效液相色谱法测定不同品种南瓜中的胡萝卜素的含量[J].华北农学报,1998,13(3):141-144.
[48]潘洪志,邱向红,李辉.南瓜提取物对小鼠S-180移植性肿瘤的影响[J].实用预防医学,2005,12(4):745-747.
[49]孔庆胜,蒋滢.南瓜多糖的组成及摩尔比测定[J].中国现代应用药学,2000,17(2):138-140.
[50]周宝兰.南瓜籽含油量及其脂肪酸组成研究[J].粮食与油脂,1997,10(1):43-45.
[51] Xanthopoulou M N, Nomikos T, Fragopoulou E, et al. Antioxidant and lipoxygenaseinhibitory activities of pumpkin seed extracts[J]. Food Research International,2009,42(5-6):641-646.
[52]尹起范,阚玉和.长白山四籽中还原糖和蛋白质的测定[J].延边大学学报,1998,24(3):28-30.
[53]范文秀,李新峥,荆瑞俊.南瓜中微量元素测定的研究[J].光谱学与光谱分析,2006,26(3):567-570.
[54]张芳,蒋作明,章恩明.南瓜的功能特性及其在食品工业中的应用[J].食品工业科技,2000,21(6):62-64.
[55]原龙,王新,杨平.大蒜中超氧化物歧化酶提取工艺的研究[J].西安工程大学学报,2009,23(1):71-77.
[56] Nedeva T, Dolashka-Angelova P, Moshtanska V, et al. Analytical technologies in thebiomedical and life sciences[J]. Journal of Chromatography B,2009,877(29):3529-3536.
[57] Dolashka-Angelova P, Angelova M, Genova L, et al. A novel Cu,Zn superoxide dismutasefrom the fungal strain Humicola lutea110: isolation and physico-chemicalcharacterization[J]. Spectrochimica Acta Part A: Molecular and BiomolecularSpectroscopy,1999,55(11):2249-2260.
[58]黄海兰,徐波,曲荣君,等. CMC-Na/DETA-B62型蛇笼树脂对金属离子的吸附性能[J].离子交换与吸附,2002,18(3):217-222.
[59]黄海兰,徐波,曲荣君,等. CMC-Na/DETA-B62型蛇笼树脂对Ag+、Cr3+和Hg2+的吸附性能[J].离子交换与吸附,2003,19(1):43-48.
[60]侯贤灯,李从容,张汉萍,等.氨基硫脲新型螯合纤维素分离富集痕量钯[J].分析化学,1994,22(5):532.
[61]陈中兰,汪模辉.吡咯烷二硫代甲酸铵纤维素的表征及其填充小柱法采集大气中的铅[J].分析化学,1997,25(6):714-717.
[62]杨冬梅,徐素英,刘克庭.利用废报纸合成树脂并提取废水中的铜离子[J].大连轻工业学院学报,2002,21(2):99-101.
[63]杨冬梅,徐素英,刘克庭.纤维素螯合树脂的合成及其对金离子的提取[J].辽宁工学院学报,2002,22(4):49-51.
[64]张亚辉,杨严俊.纤维素金属螯合亲和膜的制备及其对牛血清白蛋白的吸附研究[J].食品工业科技,2005,26(3):89-92.
[65]张亚辉,杨严俊.纤维素金属螯合亲和膜用于蛋清中功能性蛋白质的分离纯化[J].食品与生物技术学报,2006,25(2):42-47.
[66]陈观展,梁镰銮,钟珮珩.球状再生纤维素偕胺肟螯合树脂的制备及其性能研究[J].离子交换与吸附,1992,8(2):111-116.
[67] Wu C Y, Suen S Y, Chen S C, et al. A nalysis of protein adsorption on regeneratedcellulose-based immobilized copper ion affinity membranes[J]. Journal ofChromatography A,2003,996:53-70.
[68]王瑀,王丹,商士斌,等.纤维素基重金属离子吸附材料研究进展[J].生物质化学工程,2007,41(1):49-54.
[69]董绮功,张军平,李亚荣,等.新型含氮、硫纤维素螯合树脂的合成及其吸附性能[J].高等学校化学学报,2003,24(4):719-723.
[70]陈中兰. MBM新型螯合纤维素富集原子吸收测定痕量铅、镉、铜和镍[J].光谱学与光谱分析,1998,18(5):601-605.
[71]苏庆平,廖世英.硫脲型螯合纤维素对铜吸附性能的研究[J].纤维素科学与技术,1994,2(2):28-35.
[72]徐滢波,赵树进,郭勇.超氧化物歧化酶检测方法评述[J].广东药学,2002,12(1):9-12.
[73]许雅娟.邻苯三酚自氧化法测定超氧化物歧化酶活性的研究[J].西南民族大学学报(自然科学版),2006,32(6):1207-1212.
[74]袁勤生,王志友,翁清清.超氧物歧化酶的活力测定—肾上腺素自氧化法[J].中国生化药物杂志,1983,(3):4-7.
[75]林素英,吴新.四氮唑蓝(NBT)法检测SOD条件的探讨[J].海峡药学,2009,21(5):43-46.
[76]文军,葛超. NBT光还原法、邻苯三酚自氧化法测定SOD酶活性的比较[J].河北职业技术师范学院学报,2000,14(2):68-70.
[77]刘智峰,方允中.血液中超氧化物歧化酶测定的光化学扩增法[J].军事医学科学院院刊,1986,10(4):313-316.
[78]李景存,张景云.血液中SOD的化学发光测定法[J].上海医学检验杂志,1992,7(4):223-225.
[79] Dolashka-Angelova P, Stevanovic S, Dolashki A, et al. Structural and functional analysisof glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea103[J].Biochemical and Biophysical Research Communications,2004,317:1006-1016.
[80]魏文树,曾昭全,刘锡钧.超氧化物歧化酶模拟物研究进展[J].药物生物技术,2003,10(4):256-260.
[81]丁书茂,杨旭.超氧化物歧化酶及其模拟化合物研究进展[J].高等函授学报(自然科学版),2004,17(1):1-5.
[82]梅光泉.金属酶及其化学模拟[J].襄樊职业技术学院学报,2004,3(6):1-4.
[83]刘波,潘志权.锰酶及其模拟物研究进展[J].武汉化工学院学报,2004,26(4):5-13.
[84]吕蔡,张杰.槲皮素的药理作用[J].国外医药-植物药分册,2005,20(3):109-112.
[85]马文强,冯杰,何军豪.槲皮素的生物学效应及应用前景[J].饲料工业,2007,28(8):57-59.
[86]谢梅林,顾振伦,钱增年.槲皮素的抗活性氧作用[J].中国野生植物资源,1997,16(2):1-4.
[87]苏俊峰,郭长江.食物黄酮槲皮素的抗氧化作用[J].解放军预防医学杂志,2001,19(3):229-231.
[88]贾小燕,闫素清,柴保臣,等.槲皮素-钼配合物对超氧阴离子和羟自由基的清除作用[J].华西药学杂志,2008,23(1):40-42.
[89]张淑敏,郝春香.槲皮素-铁配合物的光度法研究[J].光谱实验室,2001,18(3):328-331.
[90]张淑敏,郝春香.钼-槲皮素配合物的光度法研究[J].光谱实验室,2000,17(3):333-336.
[91]余琳,黄春霞.槲皮素-铝(Ⅲ)-Tween-80荧光体系的研究及其应用[J].广东工业大学学报,2001,18(2):81-84.
[92]潘湛昌,黄宝华,刘千钧.槲皮素-锆（IV）-柠檬酸-十二烷基磺酸钠体系荧光光度法测定槲皮素的研究[J].化学世界,2001,42(10):517-519.
[93]戚琦,李蕾,罗国添.槲皮素与稀土Nd3+、Dy3+配合物合成及光谱表征[J].赣南师范学院学报,2004,25(3):5-6.
[94] Bukharia S, Memonb S, Mahroof T S, et al. Synthesis, characterization and antioxidantactivity copper-quercetin complex[J]. Spectrochimica Acta Part A,2009,71(5):1901-1906.
[95]谭君.槲皮素合铜(Ⅱ)配合物的配位化学研究[J].重庆教育学院学报,2006,19(6):11-12.
[96]夏玉明,朴惠善,许妍姬,等.槲皮素过渡金属锌配合物的合成[J].延边大学学报,1999,25(1):37-39.
[97]夏玉明,朴惠善,罗惠善.槲皮素铜配合物的合成及其活性研究[J].延边大学学报,2000,26(1):72-73.
[98]吴春,黄梅桂.槲皮素-锌(II)配合物体内外抑制亚硝胺合成的研究[J].食品科学,2007,28(9):35-38.
[99] Bukhari S B, Memon S, Tahir M, et al. Synthesis, characterization and investigation ofantioxidant activity of cobalt-quercetin complex[J]. Journal of Molecular Structure,2008,892(1-3):39-46.
[100]余燕影,俞梅兰,曹树稳.槲皮素铬(III)配合物合成及清除自由基活性研究[J].食品科学,2006,27(10):29-31.
[101]林天乐,严宝珍,胡高飞. Al(Ⅲ)-槲皮素配合物的光谱分析[J].分析化学研究简报,2006,34(8):1125-1128.
[102] Rubens F V, Souza D, Wagner F, et al. Synthesis, spectral and electrochemical propertiesof Al(III) and Zn(II) complexes with flavonoids[J]. Spectrochimica Acta Part A,2005,61(9):1985-1990.
[103]周晶,王进义,唐宁.槲皮素与金属Cd(Ⅱ)Sc(Ⅲ)配合物的合成及抗氧性研究[J].兰州大学学报,2001,37(1):123-125.
[104]陈旭龙,杨晓占,李瑞霞,等.槲皮素-Sn(II)配合物荧光、紫外及红外光谱的测量与分析[J].光散射学报,2007,19(4):369-373.
[105]余琳,吴晓桦.槲皮素-镓(Ⅲ)-十二烷基磺酸钠荧光体系及其在测定中药槐米、银杏叶、陈皮中黄酮的应用[J].理化检验-化学分册,2007,43(8):632-634.
[106]姚胜昆,赫春香.锗(Ⅵ)-槲皮素配合物的分光光度法研究[J].光谱实验室,2008,25(4):541-544.
[107] Chen W J, Sun S F, Cao W, et al. Antioxidant property of quercetin-Cr(III) complex: Therole of Cr(III) ion[J]. Journal of Molecular Structure,2009,918(1-3):194-197.
[108] Torreggiani A, Tamba M, Trinchero A, et al. Copper(II)-Quercetin complexes in aqueoussolutions: spectroscopic and kinetic properties[J]. Journal of Molecular Structure,2005,744-747:759-766.
[109] Nest G L, Caille O, Woudstra M, et al. Zn-polyphenol chelation: complexes with quercetin,(+)-catechin, and derivatives: I optical and NMR studies[J]. Inorganica Chimica Acta,2004,357(3):775-784.
[110]冯敏杰,周惠明,钱海峰.鹅血中转铁蛋白质的分离纯化及其性质[J].无锡轻工大学学报,2004,23(1):94-98.
[111]魏琪,姚汝华,鲍时翔.两种固定化金属螯合复合亲和膜色谱介质制备[J].功能高分子学报,2000,13(1):90.
[112]候宪玉,冯佑民,张友尚.转铁蛋白结构与功能的研究—鸭血清转铁蛋白含单一铁结合部位的结构域的制备和鉴定[J].生物化学杂志,1988,4(3):244-251.
[113]薛连生,郑家驹.光化增强法检测超氧化物歧化酶的初步研究[J].中华医学检验杂志,1988,11(6):329-331.
[114] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantitiesof protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.
[115] Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head ofBacteriophage T4[J]. Nature1970,227:680-685.
[116]陈晓梅,刘雅文,程熠,等.考马斯亮蓝法蛋白定量标准曲线稳定性观察[J].中国公共卫生,2006,22(3):380-381.
[117]鲍忠剑,田文儒,田昌林.热变性法提纯猪血SOD最适条件研究[J].动物医学进展,2007,28(3):20-22.
[118]吕淑霞.基础生化实验指导[M].北京:中国农业大学出版社,2003,117-118.
[119]王爱国,罗广华,邵从本,等.大豆种子超氧化物歧化酶的研究[J].植物生理学报,1983,9(1):77-84.
[120]程光宇,魏锦城,邹玉珍,等.枸杞果实铜锌超氧化物歧化酶的纯化及其性质的研究[J].南京师范大学学报自然科学版,1991,20(2):128-136.
[121] Colla G, Rouphael Y, Cardarelli M, et al. The effectiveness of grafting to improve alkalinitytolerance in watermelon[J]. Environmental and Experimental Botany,2010,68:283-291.
[122] Ahmed H, Schott E J, Gauthier J D, et al. Superoxide dismutases from the oyster parasitePerkinsus marinus: purification, biochemical characterization, and development of a platemicroassay for ctivity[J]. Analytical Biochemistry,2003,318:132-141.
[123] Krumova E, Dolashki A, Pashova S, et al. Unusual location and characterization ofCu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea[J].Archives of Microbiology,2008,189:121-130.
[124] Krumova E, Dolashka-Angelova P, Pashova S, et al. Improved production by fed-batchcultivation and some properties of Cu/Zn-superoxide dismutase from the fungal strainHumicola lutea103[J]. Enzyme and Microbial Technology,2007,40:524-532
[125] Wang Z, He Z, Li S, et al. Purification and partial characterization of Cu, Zn containingsuperoxide dismutase from entomogenous fungal species Cordyceps militaris. Enzyme andMicrobial Technology,2005,36:862-869.
[126] Ragusa S, Cambria M T, Scarpa M, et al. Properties of Purified Cytosolic Isoenzyme I ofCu,Zn-Superoxide Dismutase from Nicotiana plumbaginifolia Leaves[J]. ProteinExpression and Purification,2001,23:261-269.
[127]张廷华,李珍花,罗福明,等.外源牲超氧化物歧化酶对大鼠脑缺血再灌注保护作用的实验研究[J].卫生职业教育,2010,28(1):121-123.
[128] Corvo M L, Jorge J C S, van’t Hof R, et al. Superoxide dismutase entrapped inlong-circulating liposomes: formulation design and therapeutic activity in rat adjuvantarthritis [J]. Biochimica et Biophysica Acta,2002,1564:227-236.
[129] Liu J, Zhao T, Tan H, et al. Pharmacokinetic analysis of in vivo disposition ofheparin-superoxide dismutase [J]. Biomedicine&Pharmacotherapy,2010,64:686-691.
[130]吴云,袁勤生,顾璆.修饰超氧化物歧化酶的制备、性质及代谢[J].华东理工大学学报,1988,14(6):663-667.
[131] Angelova M, Dolashka-Angelova P, Ivanova E, et al. A novel glycosylatedCu/Zn-containing superoxide dismutase: production and potential therapeutic effect[J].Microbiology,2001,147:1641-1650.
[132] Dolashka-Angelova P, Moshtanska V, Kujumdzieva A, et al. Structure of glycosylatedCu/Zn-superoxide dismutase from Kluyveromyces yeast NBIMCC1984[J]. Journal ofMolecular Structure,2010,980:18-23.
[133]郭崇武,易胜飞.三价铬镀液中铜杂质的分析[J].电镀与精饰,2008,30(6):34-36.
[134]杨润萍,李晓霞,丁磊,等.污染水中铜离子浓度的快速测定[J].中国卫生检验杂志,2007,17(12):2217-2218.
[135]张丽萍,唐先洪,李伟挥,等.茶水中锌含量分析方法研究[J].中国卫生检验杂志,2007,17(9):1542-1543.
[2] Gomme P T, McCann K B, Bertolini J. Transferrin: structure, function and potentialtherapeutic actions[J]. Drug Discovery Today,2005,10(4):267-273.
[3]王丛珠,王登平.转铁蛋白介导作用的应用进展[J].放射免疫学杂志,2003,16(5):304-306.
[4]章华础,肖祥熊.转铁蛋白放射免疫分析的临床应用[J].放射免疫学杂志,1997,10(6):370-373.
[5]廖继佩.乳铁蛋白Lf抗病毒感染研究进展[J].国外医学病毒学分册,2003,10(6):180-183.
[6]张宏伟,刘俊涛,王晓航.转铁蛋白和转铁蛋白受体的研究进展[J].国外医学临床生物化学与检验学手册,2000,21(4):183-184.
[7] Gupta R, Rastogi S, Nagar R, et al. Dietary and serum iron, body iron stores and coronaryheart disease [J]. The Journal of the Association of Physicians of India,2000,48(5):489-492.
[8] Harris W R, Messori L. A comparative study of aluminum(III), gallium(III), indium(III),and thallium(III) binding to human serum transferring[J]. Coordination Chemistry Reviews,2002,228(2):237-262.
[9] Messori L, Orioli P, Banholzer V, et al. Formation of titanium(IV) transferrin by reaction ofhuman serum apotransferrin with titanium complexes[J]. FEBS Letters,1999,442(2-3):157-161.
[10] Sadava D, Phillips T, Lin C, et al. Transferrin overcomes drug resistance to artemisinin inhuman small-cell lung carcinoma cells[J]. Cancer Letters,2002,179(2):151-156.
[11]冯佑民,孙嘉琳,童春香,等.转铁蛋白结构与功能的研究Ⅰ.猪转铁蛋白的分离纯化及在无血清细胞培养中的应用[J].生物化学与生物物理学报,1987,19(4):322-327.
[12] Ii I, Kimura I, Ozawa E. A myotrophic protein from chick embryo extract: Its purification,identity to transferrin, and indispensability for avian myogenesis[J]. DevelopmentalBiology,1982,94(2):366-377.
[13]王革新,孙南翔,王者鹏,等.人血浆转铁蛋白的分离与纯制[J].化学试剂,1991,13(4):244-247.
[14] McCann K B, Hughes B, Wu J, et al. Purification of transferrin from Cohn supernatant Iusing ion-exchange chromatography[J]. Biotechnology And Applied Biochemistry,2005,42(3):211-217.
[15] Chung M C-M, Chan S-L, Sfflmzu S. Purification of transferrins and lactoferrin usingDEAE Affi-Gel blue[J]. International Journal of Biochemistry,1987,23(5-6):609-616.
[16] Winzerling J J, Pham D Q-D, Kunz S, et al. Purification of Recombinant Insect Transferrinfrom Large Volumes of Cell Culture Medium Using High Capacity Ni2+-DipicolylamineGel[J]. Protein Expression and Purification,1996,7(2):137-142.
[17] Jain S, Gupta M N. An integrated process for separation of major and minor proteins fromgoat serum[J]. Applied Biochemistry and Biotechnology,2005,125(1):53-62.
[18] Ardehali R, Shi L, Janatova J, et al. The inhibitory activity of serum to prevent bacterialadhesion is mainly due to apo-transferrin[J]. Journal of Biomedical Materials ResearchPart A,2003,66(1):21-28.
[19] Lim H J, Kim Y K, Hwang D S, et al. Expression of functional human transferrin in stablytransfected Drosophila S2cells[J]. Biotechnology Progress,2004,20(4):1192-1197.
[20] McCord J M, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein(hemocuprein)[J]. Journal of Biological Chemistry,1969,244(22):6049-6055.
[21] Harman D. Aging: a theory based on free radical and radiation chemistry[J]. Journal ofGerontology,1956,11(3):298-300.
[22] Van Camp W, Inzé D, Van Montagu M. The regulation and function of tobacco superoxidedismutases [J]. Free Radical Biology Medicine,1997,23(3):515-520.
[23] Fester T, Schuster W. Potato mitochondrial manganese superoxide dismutase is anRNA-binding protein[J]. Biochemistry and Molecular Biology International,1995,36(1):67-75.
[24] Parker M W, Blake C C. Iron-and manganese-containing superoxide dismutases can bedistinguished by analysis of their primary structures[J]. FEBS Letters,1988,229(2):377-382.
[25]袁勤生.超氧化物歧化酶[M].上海：华东理工大学出版社，2009,170-192.
[26] Youn H D, Kim E J, Roe J H, et al. A novel nickel-containing superoxide dismutase fromstreptomyces spp[J]. Journal of Biochemistry,1996,318:889-896.
[27] Skarpanska-Stejnborn A, Pilaczynska-Szczesniak L, Basta P, et al. Effects of OralSupplementation With Plant Superoxide Dismutase Extract on Selected Redox Parametersand an Inflammatory Marker in a2,000-m Rowing-Ergometer Test. International Journalof Sport Nutrition and Exercise Metabolism,2011,124-134.
[28] Van Camp W, Capian K, Van Montagu M, et al. Enhancement of oxidative stress tolerancein transgenic tobacco plants over producing Fe superoxide dismutase in chlorop lasts [J].Plant Physiology,1996,112(4):1703-1714.
[29]赵晓瑜,宋小青,刘建荣.玉米Mn-SOD的分离纯化与性质分析[J].河北大学学报(自然科学版),2009,29(3):324-328.
[30]宫霞,孙淑斌,孔凡华.小白菜中超氧化物歧化酶的提取及稳定性[J].曲阜师范大学学报,1997,23(1):97-99.
[31] Wang J, Liu J, Lu J, et al. Isolation and purification of superoxide dismutase from garlicusing two-stage ultrafiltration[J]. Journal of Membrane Science,2010,352(1-2):231-238.
[32] He N, Li Q B, Sun D H, et al. Isolation, purification and characterization of superoxidedismutase from garlic[J]. Biochemical Engineering Journal,2008,38(1):33-38.
[33] Sheng L Q, Tong H W, Zheng X Y, et al. Purification and characterization of cytosolicisoenzyme III of Cu,Zn-superoxide dismutase from tobacco leaves[J]. Plant Science,2004,167(6):1235-1241.
[34]王中凤,肖厚荣,杨红,等.仙人掌SOD提取条件及提取过程活性稳定性研究[J].食品与发酵工业,2008,34(7):156-158.
[35] Haddad N I A, Yuan Q. Purification and some properties of Cu,Zn superoxide dismutasefrom Radix lethospermi seed, kind of Chinese traditional medicine[J]. Journal ofChromatography B,2005,818(2):123-131
[36] Vyas D, Kumar S. Purification and partial characterization of a low temperature responsiveMn-SOD from tea (Camellia sinensis (L.) O. Kuntze)[J]. Biochemical and BiophysicalResearch Communications,2005,329(3):831-838.
[37] Kumar S, Malhotra S P. Partial purification of superoxide dismutase and peroxidase fromber (Zizyphus mauritiana Lamk.) fruit using anion exchange chromatography[J].Physiology and Molecular Biology of Plants,2008,14(3):167-172.
[38] Kochhar S, Kochhar V. Identification and characterization of a super-stable Cu–Zn SODfrom leaves of turmeric (Curcuma longal)[J]. Planta,2008,228(2):307-318
[39] Babitha M.P, Prakash H.S, et al. Purification and partial characterization of manganesesuperoxide dismutase from downy mildew resistant pearl millet seedlings inoculated withSclerospora graminicola[J]. Plant Science,2002,163(4):917-924.
[40]毛绍春,李竹英,李聪.酸果蔓中SOD酶的提取及活性测定[J].食品与机械,2007,23(1):26-46.
[41]区炳庆,何丽烂,任吉君.南瓜种子超氧化物歧化酶某些性质的研究[J].广西植物,2004,24(5):463-465.
[42]卢运超,黄兆峰.南瓜粉冲剂的研制及临床应用[J].时珍国药研究,1997,8(3):264-265.
[43]吴素玲,孙晓明,金敬宏.南瓜品质资源的分析测试[J].中国野生植物资源,2002,21(2):53-54.
[44]王燕,车振明.南瓜的功能特性及其深加工[J].食品研究与开发,2005,26(3):7-10.
[45]卢颖,王永勤,任智捷.南瓜功能成分研究的进展及在医药领域中的应用[J].食品与药品,2005,7(7A):29-32.
[46]张建农,满艳萍.南瓜果实营养成分测定与分析[J].甘肃农大学学报1994,34(3):300-302.
[47]吴增茹,金同铭.用高效液相色谱法测定不同品种南瓜中的胡萝卜素的含量[J].华北农学报,1998,13(3):141-144.
[48]潘洪志,邱向红,李辉.南瓜提取物对小鼠S-180移植性肿瘤的影响[J].实用预防医学,2005,12(4):745-747.
[49]孔庆胜,蒋滢.南瓜多糖的组成及摩尔比测定[J].中国现代应用药学,2000,17(2):138-140.
[50]周宝兰.南瓜籽含油量及其脂肪酸组成研究[J].粮食与油脂,1997,10(1):43-45.
[51] Xanthopoulou M N, Nomikos T, Fragopoulou E, et al. Antioxidant and lipoxygenaseinhibitory activities of pumpkin seed extracts[J]. Food Research International,2009,42(5-6):641-646.
[52]尹起范,阚玉和.长白山四籽中还原糖和蛋白质的测定[J].延边大学学报,1998,24(3):28-30.
[53]范文秀,李新峥,荆瑞俊.南瓜中微量元素测定的研究[J].光谱学与光谱分析,2006,26(3):567-570.
[54]张芳,蒋作明,章恩明.南瓜的功能特性及其在食品工业中的应用[J].食品工业科技,2000,21(6):62-64.
[55]原龙,王新,杨平.大蒜中超氧化物歧化酶提取工艺的研究[J].西安工程大学学报,2009,23(1):71-77.
[56] Nedeva T, Dolashka-Angelova P, Moshtanska V, et al. Analytical technologies in thebiomedical and life sciences[J]. Journal of Chromatography B,2009,877(29):3529-3536.
[57] Dolashka-Angelova P, Angelova M, Genova L, et al. A novel Cu,Zn superoxide dismutasefrom the fungal strain Humicola lutea110: isolation and physico-chemicalcharacterization[J]. Spectrochimica Acta Part A: Molecular and BiomolecularSpectroscopy,1999,55(11):2249-2260.
[58]黄海兰,徐波,曲荣君,等. CMC-Na/DETA-B62型蛇笼树脂对金属离子的吸附性能[J].离子交换与吸附,2002,18(3):217-222.
[59]黄海兰,徐波,曲荣君,等. CMC-Na/DETA-B62型蛇笼树脂对Ag+、Cr3+和Hg2+的吸附性能[J].离子交换与吸附,2003,19(1):43-48.
[60]侯贤灯,李从容,张汉萍,等.氨基硫脲新型螯合纤维素分离富集痕量钯[J].分析化学,1994,22(5):532.
[61]陈中兰,汪模辉.吡咯烷二硫代甲酸铵纤维素的表征及其填充小柱法采集大气中的铅[J].分析化学,1997,25(6):714-717.
[62]杨冬梅,徐素英,刘克庭.利用废报纸合成树脂并提取废水中的铜离子[J].大连轻工业学院学报,2002,21(2):99-101.
[63]杨冬梅,徐素英,刘克庭.纤维素螯合树脂的合成及其对金离子的提取[J].辽宁工学院学报,2002,22(4):49-51.
[64]张亚辉,杨严俊.纤维素金属螯合亲和膜的制备及其对牛血清白蛋白的吸附研究[J].食品工业科技,2005,26(3):89-92.
[65]张亚辉,杨严俊.纤维素金属螯合亲和膜用于蛋清中功能性蛋白质的分离纯化[J].食品与生物技术学报,2006,25(2):42-47.
[66]陈观展,梁镰銮,钟珮珩.球状再生纤维素偕胺肟螯合树脂的制备及其性能研究[J].离子交换与吸附,1992,8(2):111-116.
[67] Wu C Y, Suen S Y, Chen S C, et al. A nalysis of protein adsorption on regeneratedcellulose-based immobilized copper ion affinity membranes[J]. Journal ofChromatography A,2003,996:53-70.
[68]王瑀,王丹,商士斌,等.纤维素基重金属离子吸附材料研究进展[J].生物质化学工程,2007,41(1):49-54.
[69]董绮功,张军平,李亚荣,等.新型含氮、硫纤维素螯合树脂的合成及其吸附性能[J].高等学校化学学报,2003,24(4):719-723.
[70]陈中兰. MBM新型螯合纤维素富集原子吸收测定痕量铅、镉、铜和镍[J].光谱学与光谱分析,1998,18(5):601-605.
[71]苏庆平,廖世英.硫脲型螯合纤维素对铜吸附性能的研究[J].纤维素科学与技术,1994,2(2):28-35.
[72]徐滢波,赵树进,郭勇.超氧化物歧化酶检测方法评述[J].广东药学,2002,12(1):9-12.
[73]许雅娟.邻苯三酚自氧化法测定超氧化物歧化酶活性的研究[J].西南民族大学学报(自然科学版),2006,32(6):1207-1212.
[74]袁勤生,王志友,翁清清.超氧物歧化酶的活力测定—肾上腺素自氧化法[J].中国生化药物杂志,1983,(3):4-7.
[75]林素英,吴新.四氮唑蓝(NBT)法检测SOD条件的探讨[J].海峡药学,2009,21(5):43-46.
[76]文军,葛超. NBT光还原法、邻苯三酚自氧化法测定SOD酶活性的比较[J].河北职业技术师范学院学报,2000,14(2):68-70.
[77]刘智峰,方允中.血液中超氧化物歧化酶测定的光化学扩增法[J].军事医学科学院院刊,1986,10(4):313-316.
[78]李景存,张景云.血液中SOD的化学发光测定法[J].上海医学检验杂志,1992,7(4):223-225.
[79] Dolashka-Angelova P, Stevanovic S, Dolashki A, et al. Structural and functional analysisof glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea103[J].Biochemical and Biophysical Research Communications,2004,317:1006-1016.
[80]魏文树,曾昭全,刘锡钧.超氧化物歧化酶模拟物研究进展[J].药物生物技术,2003,10(4):256-260.
[81]丁书茂,杨旭.超氧化物歧化酶及其模拟化合物研究进展[J].高等函授学报(自然科学版),2004,17(1):1-5.
[82]梅光泉.金属酶及其化学模拟[J].襄樊职业技术学院学报,2004,3(6):1-4.
[83]刘波,潘志权.锰酶及其模拟物研究进展[J].武汉化工学院学报,2004,26(4):5-13.
[84]吕蔡,张杰.槲皮素的药理作用[J].国外医药-植物药分册,2005,20(3):109-112.
[85]马文强,冯杰,何军豪.槲皮素的生物学效应及应用前景[J].饲料工业,2007,28(8):57-59.
[86]谢梅林,顾振伦,钱增年.槲皮素的抗活性氧作用[J].中国野生植物资源,1997,16(2):1-4.
[87]苏俊峰,郭长江.食物黄酮槲皮素的抗氧化作用[J].解放军预防医学杂志,2001,19(3):229-231.
[88]贾小燕,闫素清,柴保臣,等.槲皮素-钼配合物对超氧阴离子和羟自由基的清除作用[J].华西药学杂志,2008,23(1):40-42.
[89]张淑敏,郝春香.槲皮素-铁配合物的光度法研究[J].光谱实验室,2001,18(3):328-331.
[90]张淑敏,郝春香.钼-槲皮素配合物的光度法研究[J].光谱实验室,2000,17(3):333-336.
[91]余琳,黄春霞.槲皮素-铝(Ⅲ)-Tween-80荧光体系的研究及其应用[J].广东工业大学学报,2001,18(2):81-84.
[92]潘湛昌,黄宝华,刘千钧.槲皮素-锆（IV）-柠檬酸-十二烷基磺酸钠体系荧光光度法测定槲皮素的研究[J].化学世界,2001,42(10):517-519.
[93]戚琦,李蕾,罗国添.槲皮素与稀土Nd3+、Dy3+配合物合成及光谱表征[J].赣南师范学院学报,2004,25(3):5-6.
[94] Bukharia S, Memonb S, Mahroof T S, et al. Synthesis, characterization and antioxidantactivity copper-quercetin complex[J]. Spectrochimica Acta Part A,2009,71(5):1901-1906.
[95]谭君.槲皮素合铜(Ⅱ)配合物的配位化学研究[J].重庆教育学院学报,2006,19(6):11-12.
[96]夏玉明,朴惠善,许妍姬,等.槲皮素过渡金属锌配合物的合成[J].延边大学学报,1999,25(1):37-39.
[97]夏玉明,朴惠善,罗惠善.槲皮素铜配合物的合成及其活性研究[J].延边大学学报,2000,26(1):72-73.
[98]吴春,黄梅桂.槲皮素-锌(II)配合物体内外抑制亚硝胺合成的研究[J].食品科学,2007,28(9):35-38.
[99] Bukhari S B, Memon S, Tahir M, et al. Synthesis, characterization and investigation ofantioxidant activity of cobalt-quercetin complex[J]. Journal of Molecular Structure,2008,892(1-3):39-46.
[100]余燕影,俞梅兰,曹树稳.槲皮素铬(III)配合物合成及清除自由基活性研究[J].食品科学,2006,27(10):29-31.
[101]林天乐,严宝珍,胡高飞. Al(Ⅲ)-槲皮素配合物的光谱分析[J].分析化学研究简报,2006,34(8):1125-1128.
[102] Rubens F V, Souza D, Wagner F, et al. Synthesis, spectral and electrochemical propertiesof Al(III) and Zn(II) complexes with flavonoids[J]. Spectrochimica Acta Part A,2005,61(9):1985-1990.
[103]周晶,王进义,唐宁.槲皮素与金属Cd(Ⅱ)Sc(Ⅲ)配合物的合成及抗氧性研究[J].兰州大学学报,2001,37(1):123-125.
[104]陈旭龙,杨晓占,李瑞霞,等.槲皮素-Sn(II)配合物荧光、紫外及红外光谱的测量与分析[J].光散射学报,2007,19(4):369-373.
[105]余琳,吴晓桦.槲皮素-镓(Ⅲ)-十二烷基磺酸钠荧光体系及其在测定中药槐米、银杏叶、陈皮中黄酮的应用[J].理化检验-化学分册,2007,43(8):632-634.
[106]姚胜昆,赫春香.锗(Ⅵ)-槲皮素配合物的分光光度法研究[J].光谱实验室,2008,25(4):541-544.
[107] Chen W J, Sun S F, Cao W, et al. Antioxidant property of quercetin-Cr(III) complex: Therole of Cr(III) ion[J]. Journal of Molecular Structure,2009,918(1-3):194-197.
[108] Torreggiani A, Tamba M, Trinchero A, et al. Copper(II)-Quercetin complexes in aqueoussolutions: spectroscopic and kinetic properties[J]. Journal of Molecular Structure,2005,744-747:759-766.
[109] Nest G L, Caille O, Woudstra M, et al. Zn-polyphenol chelation: complexes with quercetin,(+)-catechin, and derivatives: I optical and NMR studies[J]. Inorganica Chimica Acta,2004,357(3):775-784.
[110]冯敏杰,周惠明,钱海峰.鹅血中转铁蛋白质的分离纯化及其性质[J].无锡轻工大学学报,2004,23(1):94-98.
[111]魏琪,姚汝华,鲍时翔.两种固定化金属螯合复合亲和膜色谱介质制备[J].功能高分子学报,2000,13(1):90.
[112]候宪玉,冯佑民,张友尚.转铁蛋白结构与功能的研究—鸭血清转铁蛋白含单一铁结合部位的结构域的制备和鉴定[J].生物化学杂志,1988,4(3):244-251.
[113]薛连生,郑家驹.光化增强法检测超氧化物歧化酶的初步研究[J].中华医学检验杂志,1988,11(6):329-331.
[114] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantitiesof protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry,1976,72:248-254.
[115] Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head ofBacteriophage T4[J]. Nature1970,227:680-685.
[116]陈晓梅,刘雅文,程熠,等.考马斯亮蓝法蛋白定量标准曲线稳定性观察[J].中国公共卫生,2006,22(3):380-381.
[117]鲍忠剑,田文儒,田昌林.热变性法提纯猪血SOD最适条件研究[J].动物医学进展,2007,28(3):20-22.
[118]吕淑霞.基础生化实验指导[M].北京:中国农业大学出版社,2003,117-118.
[119]王爱国,罗广华,邵从本,等.大豆种子超氧化物歧化酶的研究[J].植物生理学报,1983,9(1):77-84.
[120]程光宇,魏锦城,邹玉珍,等.枸杞果实铜锌超氧化物歧化酶的纯化及其性质的研究[J].南京师范大学学报自然科学版,1991,20(2):128-136.
[121] Colla G, Rouphael Y, Cardarelli M, et al. The effectiveness of grafting to improve alkalinitytolerance in watermelon[J]. Environmental and Experimental Botany,2010,68:283-291.
[122] Ahmed H, Schott E J, Gauthier J D, et al. Superoxide dismutases from the oyster parasitePerkinsus marinus: purification, biochemical characterization, and development of a platemicroassay for ctivity[J]. Analytical Biochemistry,2003,318:132-141.
[123] Krumova E, Dolashki A, Pashova S, et al. Unusual location and characterization ofCu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea[J].Archives of Microbiology,2008,189:121-130.
[124] Krumova E, Dolashka-Angelova P, Pashova S, et al. Improved production by fed-batchcultivation and some properties of Cu/Zn-superoxide dismutase from the fungal strainHumicola lutea103[J]. Enzyme and Microbial Technology,2007,40:524-532
[125] Wang Z, He Z, Li S, et al. Purification and partial characterization of Cu, Zn containingsuperoxide dismutase from entomogenous fungal species Cordyceps militaris. Enzyme andMicrobial Technology,2005,36:862-869.
[126] Ragusa S, Cambria M T, Scarpa M, et al. Properties of Purified Cytosolic Isoenzyme I ofCu,Zn-Superoxide Dismutase from Nicotiana plumbaginifolia Leaves[J]. ProteinExpression and Purification,2001,23:261-269.
[127]张廷华,李珍花,罗福明,等.外源牲超氧化物歧化酶对大鼠脑缺血再灌注保护作用的实验研究[J].卫生职业教育,2010,28(1):121-123.
[128] Corvo M L, Jorge J C S, van’t Hof R, et al. Superoxide dismutase entrapped inlong-circulating liposomes: formulation design and therapeutic activity in rat adjuvantarthritis [J]. Biochimica et Biophysica Acta,2002,1564:227-236.
[129] Liu J, Zhao T, Tan H, et al. Pharmacokinetic analysis of in vivo disposition ofheparin-superoxide dismutase [J]. Biomedicine&Pharmacotherapy,2010,64:686-691.
[130]吴云,袁勤生,顾璆.修饰超氧化物歧化酶的制备、性质及代谢[J].华东理工大学学报,1988,14(6):663-667.
[131] Angelova M, Dolashka-Angelova P, Ivanova E, et al. A novel glycosylatedCu/Zn-containing superoxide dismutase: production and potential therapeutic effect[J].Microbiology,2001,147:1641-1650.
[132] Dolashka-Angelova P, Moshtanska V, Kujumdzieva A, et al. Structure of glycosylatedCu/Zn-superoxide dismutase from Kluyveromyces yeast NBIMCC1984[J]. Journal ofMolecular Structure,2010,980:18-23.
[133]郭崇武,易胜飞.三价铬镀液中铜杂质的分析[J].电镀与精饰,2008,30(6):34-36.
[134]杨润萍,李晓霞,丁磊,等.污染水中铜离子浓度的快速测定[J].中国卫生检验杂志,2007,17(12):2217-2218.
[135]张丽萍,唐先洪,李伟挥,等.茶水中锌含量分析方法研究[J].中国卫生检验杂志,2007,17(9):1542-1543.