兔血清对氧磷酶的纯化及检测
摘要
目的
对氧磷酶(paraxonase,PON)是生物体内自身产生的一种能够直接水解有机磷酸酯类物质的A2酯酶类,它能够水解有机磷酸酯、芳香族羧酸酯、氨基甲酸酯和不饱和脂肪酸等多种物质。对氧磷酶在多种生物体内均有分布,在哺乳动物中以兔血清对氧磷酶的活性最高。本实验从兔血清中提取并纯化对氧磷酶,并建立PON的活性检测方法。为进一步研究对氧磷酶的分子生物学特征及其水解有机磷类化合物的机制奠定基础。
方法
建立检测对氧磷酶活性的方法。选用对氧磷作为底物。因其在对氧磷酶的作用下可以被水解成磷酸二乙酯和对硝基酚。产物中的对硝基酚在405nm波长处有明显的吸光度峰值。据此可以采用连续监测法,检测吸光度值的变化,来换算待测样品中对氧磷酶的水解活性。
选用健康新西兰白兔10只进行采血,15～20ml/只。离心后获得混合血清100ml。再采用CaCl_2盐析沉淀的方法除杂获取兔血清提取液。
采用液相色谱技术(liquid Chromatography)从兔血清中提取并纯化对氧磷酶。首先采用亲和色谱技术对血清中的蛋白质进行初步提取。汽巴兰琼脂糖-3GA3000-CL对血清脂蛋白有特异吸附作用。选择其作为亲和层析介质,对兔血清中的目的酶蛋白进行亲和液相层析。通过对色谱柱进行平衡、洗涤、洗脱等连续操作后,获得对氧磷酶的初提取液。检测其对氧磷酶活性,对酶活性高的部分进行透析除盐和浓缩脱水。
再采用离子交换液相色谱的方法对粗提取液中的不同等电点的蛋白进行分离。根据对氧磷酶的等电点IP=5.1,在条件PH值为8.0的情况下,选用DEAE-Sepharose CL-6B这种弱阴离子交换树脂为层析介质。根据对氧磷酶的疏水性,选用非离子表面活性剂使其在层析的过程中充分的乳化,令其充分的同层析柱进行弱阴离子交换吸附。通过线性递增的盐浓度梯度(0～0.35mmol/L NaCl)将其洗脱。Sepharose的胶联颗粒还具有分子筛作用,能够将兔血清中不同等电点和分子量的蛋白进一步分离开来。选择酶活性高的部分进行透析除盐和浓缩脱水达到对PON提取和纯化的目的。通过两次离子交换色谱获得物质均一的对氧磷酶。
在提纯的过程中,使用适当浓度的鳌合剂EDTA来保持Ca~(2+)离子浓度的稳定,以保证酶活性的稳定。
选用小牛血清蛋白标准溶液,采用考马斯亮蓝的方法,绘制蛋白浓度标准曲线。检测提取物的蛋白浓度。
采用SDS-PAGE垂直板电泳的方法,检测纯化后的对氧磷酶的分子量及其均一性。
结果
经检测,提纯前混合兔血清100ml的对氧磷酶的总活力为317.64U,比活性为0.058 U/mg,经三次柱层析后获得提纯后的酶蛋白约1.9ml,检测得总活力为32.31U,比活性为12.15 U/mg,是纯化前210倍左右,但产出率仅为10%左右。纯化后的酶蛋白在进行SDS-PAGE电泳后,只在43kd附近出现一条电泳带,说明纯化的效果明显。
结论
采用液相色谱的方法,可以有效地从兔血清中分离并纯化对氧磷酶。选择适当的凝胶组合可以将纯化的效果大大提高。
Objective
PON is a kind of phospholipase generated by organism itself. Current research also found that it can hydrolyze organophosphate, aromatic ester, carbamate and unsaturated fatty acid. PON widely distributes among many kinds of animals, while the PON in rabbit serum has the highest activity. This experiment is designed to derive and purify the PON in rabbit serum and establish the method to test its activity in order to make basis to furtherly explore its characteristics of molecular biology and mechanism of hydrolyzing organophosphorus compound, and to supply clew to treat organophosophorus poisoning.
Method
Establish the method to test the activity of PON. Paraoxon can be hydrolyzed by PON. Making use of that the product p-nitrophenol has absorption peak at 405nm, we can test the activity of PON by observing the change of absorbance continuously under such wave length.
Choose 10 New Zealand rabbits to acquire 100 mixed serum by centrifuge the blood acquired from them. By the method of salting out precipitation to get the crude enzyme protein.
Use the method of liquid chromatography to derive the paraoxonase in rabbit serum and purify it. Firstly derive the protein in serum by affinity chromatography.Cibacron Blue Agarose has specific affinity to lipoprotein in serum, choose it as affinity choramtography medium to absorb the paraoxonase in the serum. After the equilibrating, scrubbing and eluting, get the crude liquid of paraoxonase. Choose the parts having higher activity for dialysis and concentration。
Use the method of ion exchange chromatography to separate the protein of different isoelectric point and molecular weight. According to IP of PON is 5.1, choose the DEAE-Sepharose CL-6B as the ion exchange medium under the condition of PH=8.0. For PON is hydrophibic protein, surfactant can increase its solubility and affinity to resine during the ion exchange chromatography. Make use of its molecular seive mechanism with sodium chloride of gradient concentration to separate the protein of different IP and molecular weight.
During the purification, choose EDTA of suitable density to retain the density of Ca~(2+) so as to stablize the activity of PON.
Use the method of Commassie Brillinat Blue to draw standard curve so as to test the density of partly purified protein.
Use the method of SDS-PAG electrophoresis to determine the puritiry of partly purified paraoxonase.
Result
The result of the determination of the 100 ml mixed rabbit serum is that its total activity is 317.64 U and the specific activity of paraoxonase is 0.058 U/mg, while the specific activity is elevated to 12.15U/mg after purification, which means the purification has elevated the PON activity in rabbit serum about 210 fold . However the total activity descends to 32.13 U with the yield near 10% or so. On the SDS-PAGE electrophoresis, only one band appears near the 43 kd, which shows that the purification is of good quality.
Conclusion
Chromatography method can be used to derive and purify the paraoxonase in rabbit serum. Choosing suitable gel can greatly improved the efficacy of purification.
对氧磷酶(paraxonase,PON)是生物体内自身产生的一种能够直接水解有机磷酸酯类物质的A2酯酶类,它能够水解有机磷酸酯、芳香族羧酸酯、氨基甲酸酯和不饱和脂肪酸等多种物质。对氧磷酶在多种生物体内均有分布,在哺乳动物中以兔血清对氧磷酶的活性最高。本实验从兔血清中提取并纯化对氧磷酶,并建立PON的活性检测方法。为进一步研究对氧磷酶的分子生物学特征及其水解有机磷类化合物的机制奠定基础。
方法
建立检测对氧磷酶活性的方法。选用对氧磷作为底物。因其在对氧磷酶的作用下可以被水解成磷酸二乙酯和对硝基酚。产物中的对硝基酚在405nm波长处有明显的吸光度峰值。据此可以采用连续监测法,检测吸光度值的变化,来换算待测样品中对氧磷酶的水解活性。
选用健康新西兰白兔10只进行采血,15～20ml/只。离心后获得混合血清100ml。再采用CaCl_2盐析沉淀的方法除杂获取兔血清提取液。
采用液相色谱技术(liquid Chromatography)从兔血清中提取并纯化对氧磷酶。首先采用亲和色谱技术对血清中的蛋白质进行初步提取。汽巴兰琼脂糖-3GA3000-CL对血清脂蛋白有特异吸附作用。选择其作为亲和层析介质,对兔血清中的目的酶蛋白进行亲和液相层析。通过对色谱柱进行平衡、洗涤、洗脱等连续操作后,获得对氧磷酶的初提取液。检测其对氧磷酶活性,对酶活性高的部分进行透析除盐和浓缩脱水。
再采用离子交换液相色谱的方法对粗提取液中的不同等电点的蛋白进行分离。根据对氧磷酶的等电点IP=5.1,在条件PH值为8.0的情况下,选用DEAE-Sepharose CL-6B这种弱阴离子交换树脂为层析介质。根据对氧磷酶的疏水性,选用非离子表面活性剂使其在层析的过程中充分的乳化,令其充分的同层析柱进行弱阴离子交换吸附。通过线性递增的盐浓度梯度(0～0.35mmol/L NaCl)将其洗脱。Sepharose的胶联颗粒还具有分子筛作用,能够将兔血清中不同等电点和分子量的蛋白进一步分离开来。选择酶活性高的部分进行透析除盐和浓缩脱水达到对PON提取和纯化的目的。通过两次离子交换色谱获得物质均一的对氧磷酶。
在提纯的过程中,使用适当浓度的鳌合剂EDTA来保持Ca~(2+)离子浓度的稳定,以保证酶活性的稳定。
选用小牛血清蛋白标准溶液,采用考马斯亮蓝的方法,绘制蛋白浓度标准曲线。检测提取物的蛋白浓度。
采用SDS-PAGE垂直板电泳的方法,检测纯化后的对氧磷酶的分子量及其均一性。
结果
经检测,提纯前混合兔血清100ml的对氧磷酶的总活力为317.64U,比活性为0.058 U/mg,经三次柱层析后获得提纯后的酶蛋白约1.9ml,检测得总活力为32.31U,比活性为12.15 U/mg,是纯化前210倍左右,但产出率仅为10%左右。纯化后的酶蛋白在进行SDS-PAGE电泳后,只在43kd附近出现一条电泳带,说明纯化的效果明显。
结论
采用液相色谱的方法,可以有效地从兔血清中分离并纯化对氧磷酶。选择适当的凝胶组合可以将纯化的效果大大提高。
Objective
PON is a kind of phospholipase generated by organism itself. Current research also found that it can hydrolyze organophosphate, aromatic ester, carbamate and unsaturated fatty acid. PON widely distributes among many kinds of animals, while the PON in rabbit serum has the highest activity. This experiment is designed to derive and purify the PON in rabbit serum and establish the method to test its activity in order to make basis to furtherly explore its characteristics of molecular biology and mechanism of hydrolyzing organophosphorus compound, and to supply clew to treat organophosophorus poisoning.
Method
Establish the method to test the activity of PON. Paraoxon can be hydrolyzed by PON. Making use of that the product p-nitrophenol has absorption peak at 405nm, we can test the activity of PON by observing the change of absorbance continuously under such wave length.
Choose 10 New Zealand rabbits to acquire 100 mixed serum by centrifuge the blood acquired from them. By the method of salting out precipitation to get the crude enzyme protein.
Use the method of liquid chromatography to derive the paraoxonase in rabbit serum and purify it. Firstly derive the protein in serum by affinity chromatography.Cibacron Blue Agarose has specific affinity to lipoprotein in serum, choose it as affinity choramtography medium to absorb the paraoxonase in the serum. After the equilibrating, scrubbing and eluting, get the crude liquid of paraoxonase. Choose the parts having higher activity for dialysis and concentration。
Use the method of ion exchange chromatography to separate the protein of different isoelectric point and molecular weight. According to IP of PON is 5.1, choose the DEAE-Sepharose CL-6B as the ion exchange medium under the condition of PH=8.0. For PON is hydrophibic protein, surfactant can increase its solubility and affinity to resine during the ion exchange chromatography. Make use of its molecular seive mechanism with sodium chloride of gradient concentration to separate the protein of different IP and molecular weight.
During the purification, choose EDTA of suitable density to retain the density of Ca~(2+) so as to stablize the activity of PON.
Use the method of Commassie Brillinat Blue to draw standard curve so as to test the density of partly purified protein.
Use the method of SDS-PAG electrophoresis to determine the puritiry of partly purified paraoxonase.
Result
The result of the determination of the 100 ml mixed rabbit serum is that its total activity is 317.64 U and the specific activity of paraoxonase is 0.058 U/mg, while the specific activity is elevated to 12.15U/mg after purification, which means the purification has elevated the PON activity in rabbit serum about 210 fold . However the total activity descends to 32.13 U with the yield near 10% or so. On the SDS-PAGE electrophoresis, only one band appears near the 43 kd, which shows that the purification is of good quality.
Conclusion
Chromatography method can be used to derive and purify the paraoxonase in rabbit serum. Choosing suitable gel can greatly improved the efficacy of purification.
引文
1 Knaak JB,Dary CC,Power F,et al.Physicochemical and biological data for the development of predictive organophosphorus pesticide QSARs and PBPK/PD models for human risk assessment.Crit Rev Yoxicol 2004;34(2):143-207.
2 王汉斌.急性有机磷农药中毒救治中的突击量氯解磷定疗法[J].中国医刊 2004,(12)
3 关里,王汉斌,赵德禄.肟类复能剂治疗急性有机磷农药中毒的研究现状[J].中华内科杂志 2004,43(2):157-158
4 宁江海,王玉霞,王青定,等.人血清对氧磷酶的部分纯化及解毒效应[J].中国药理学与毒理学杂志,2002,16(1):60-65.
5 Daniel Rochu,Eric Chabriere,Patrick Masson.Human paraoxonase:A promising approach for pre-treatment and therapy of organophosphorus poisoning[J].Toxicology,2007,233(1-3):47-59
6 周志俊,邬红梅,胡云平,等血清对氧磷酶活性测定方法研究[J].中国公共卫生,2000,16(4):303-304
7 Clement E,Furlong,Rebecca et al.Purification of Rabbit and Human Serum Paraoxonaset[J].Biochemistry 1991,30,10133-10140
8 马宏伟,赵卫国,潘柏申亲和层析法分离纯化人心肌肌钙蛋白I[J]检验医学 2007,22(3):304-307
9 Karen N,Gan,Andrew Smolen,et al.Purification of human serum paraoxonase/arylesterase[J]Drug Metabolism and Disposition 1991,19(1):100-106
10 杜玮南 温进坤 韩梅.人血清对氧磷酶活性测定方法的建立及临床初步应用[J].河北医科大学学报,1999,20(3):134-136
11 邵军刘智广SDS-聚丙烯酰氨凝胶电泳检测尿激酶纯度[J]第四军医大学学报 1993,14(2):158-160
12 Reiner E.Recommendations of the IUBUM Nomenclature Committee:comments concerting classification and nomenclature of esterases hydrolysing organophosphorus compounds[J].Chem Biol Interact,1993,87(1-3):15-16.
13 Kuo CL,La Du BN.Comparison of purified human and rabbit serum paraoxonase J.Drug Metab Dispos,1995,23(9):935-944.
14 胡红焱 邹德勇 崔云龙 对氧磷酶Paraoxonase的测定方法与临床意义[J].国外医学临床生物化学与检验学分册 2001,22(2)73-76
15 赵赞梅 王汉斌 对氧磷酶的某些研究进展[J]毒理学杂志,2005,19(1):64-66
16 Hernandz A,Gonzalvo MC,Gil F,et al.Distribution profiles ofparaoxonase and choliesterase phenotypes in a Spanish population[J].Chem Biol Interact,1999,14(119-120):201-209.
17 申本昌 乔传令 对氧磷酶及其生理功能的研究进展[J]生命的化学,2002,22(3):225-227
18 CE Furlong,LG Costa,C Hassett,et al.Human and Rabbit Paraoxonase:Purification,Cloing,Sequencing,Mapping and Role of Polymorphism in Organophosophate Detoxification.[J].Chem.-Biol.Interactions,1993,87:35-48
19 Daniel Rochu,Eric Chabriere,Patrick Masson.Human paraoxonase:A promising approach for pre-treatment and therapy of organophosphorus poisoning[J].Toxicology,2007,233(1-3):47-59
20 I.Petrikovics,D.Papahadjopoulos,K.Hong,et al.Comparing Therapeutic and Prophylactic Protection against the Lethal Effect of Paraoxon[J].Toxicology Science,2004,77(2):258-262.
2]何庆,万智,唐伦.有机磷中毒救治与预防的重大进步-PON1研究进展[J].中华急诊医学杂志,2003,12(6):425-426
22 La Du BN,Adkins S,Kuo CL,et al.Studies on human serum paraoxonase/arylesterase[J].Chem Biol Interact,1993,87(1-3):25-34.
23 Mackenss B,Mackness MI,Arrol S,et al.Serum paraoxonase 55 and 192 polymorphism and paraoxonase activity and concentration in non-insulin dependent diabetets mellitus[J].Atherosclerosis.1998,139(2):341-349
24 傅强 刘秉文 血清及高密度脂蛋白中对氧磷酶活性的分光光度测定法[J].华西医大学报,2001;32 3:459-461
1 Reiner E.Recommendations of the IUBUM Nomenclature Committee:comments concening classification and nomenclature of esterases hydrolysing organophosphorus compounds[J].Chem Biol Interact,t993,87(1-3):15-16.
2 宁江海,王玉霞,王青定,等.人血清对氧磷酶的部分纯化及解毒效应[J].中国药理学与毒理学杂志,2002,16(1):60-65.
3 Hernandz A,Gonzalvo MC,Gil F,et al.Distribution profiles of paraoxonase and choliesterase phenotypes in a Spanish population[J].Chem Biol Interact,1999,14(119-120):201-209.
4 Furlong CE,Richter RJ,Chapline C,et al.Purification of rabbit and human serum paraoxonase[J].Biochemistry,1991,30(42):10133-10140.
5 周志俊,邬红梅,周建芳,等.上海市正常人群血清对氧磷酶活性及多态性分布特征[J].中华预防医学杂志,2000,34(1):20-21.
6 滕霞,孙曼霁.对氧磷酶研究进展[J].生命科学,2002,14(2):107-111.
7 Sorenson RC,Primo-parmo SL,Kuo CL,et al.Reconsideration of the Catalytic Centerand Mechanism of Mammalian Paraoxonase/Arylesterase[J].Proc Natl Acad Sci U S A,1995,92(16):7187-7191.
8 王刚垛,何凤生.对氧磷酶基因多态性及其临床意义的研究进展[J].中国工业医学杂志,1999,12(6):360-362.
9 Kuo CL,La Du BN.Comparison of Purified Human and Rabbit Serum Paraoxonases[J].Drug Metab Dispos,1995;23(9):935.
10 赵赞梅,王汉斌.对氧磷酶的某些研究进展[J].毒理学杂志,2005,19(1):64-66.
11 方定志,张朝良,刘秉文,等.人血清对氧磷酶-1 活性与血脂及主要载脂蛋白的相关性初探[J].四川大学学报,2003,34(1):162-163.
12 Daniel Rochu,Eric Chabriere,Patrick Masson.Human paraoxonase:A promising approach for pre-treatment and therapy of organophosphorus poisoning[J].Toxicology,2007,233(1-3):47-59
13 La Du BN,Adkins S,Kuo CL,et al.Studies on human serum paraoxonase/arylesterase[J].Chem Biol Interact,1993,87(1-3):25-34.
14 I.Petrikovics,D.Papahadjopoulos,K.Hong,et al.Comparing Therapeutic and Prophylactic Protection against the Lethal Effect of Paraoxon[J].Toxicology Science,2004,77(2):258-262.
15 Catherine Mee-Hie Cho,Ashok Mulchandani,Wilfred Chen.Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos[J].Applied and Environmental Microbiology,2004,70(8):4681-4685.
16 杨玉恒,马桂芬,薛永蕙.对氧磷酶-1与有机磷农药中毒的关系[J].河北医药.2005,2711):856-857.
17 Mackenss B,Mackness MI,Arrol S,et al.Serum paraoxonase 55 and 192 polymorphism and paraoxonase activity and concentration in non-insulin dependent diabetets mellitus[J].Atherosclerosis.1998,139(2):341-349
18 La Du BN.Structural and functional diversity of paraoxonases.Nature Med,1996,2(11):1186-1187.
19 王垛刚,何凤生.对氧磷酶基因多态性及其临床意义的研究进展[J].中国工业医学杂志,1999,(12),360-362
20 何庆,万智,唐伦.有机磷中毒救治与预防的重大进步-PON1研究进展[J].中华急诊医学杂志,2003,12(6):425-426
2 王汉斌.急性有机磷农药中毒救治中的突击量氯解磷定疗法[J].中国医刊 2004,(12)
3 关里,王汉斌,赵德禄.肟类复能剂治疗急性有机磷农药中毒的研究现状[J].中华内科杂志 2004,43(2):157-158
4 宁江海,王玉霞,王青定,等.人血清对氧磷酶的部分纯化及解毒效应[J].中国药理学与毒理学杂志,2002,16(1):60-65.
5 Daniel Rochu,Eric Chabriere,Patrick Masson.Human paraoxonase:A promising approach for pre-treatment and therapy of organophosphorus poisoning[J].Toxicology,2007,233(1-3):47-59
6 周志俊,邬红梅,胡云平,等血清对氧磷酶活性测定方法研究[J].中国公共卫生,2000,16(4):303-304
7 Clement E,Furlong,Rebecca et al.Purification of Rabbit and Human Serum Paraoxonaset[J].Biochemistry 1991,30,10133-10140
8 马宏伟,赵卫国,潘柏申亲和层析法分离纯化人心肌肌钙蛋白I[J]检验医学 2007,22(3):304-307
9 Karen N,Gan,Andrew Smolen,et al.Purification of human serum paraoxonase/arylesterase[J]Drug Metabolism and Disposition 1991,19(1):100-106
10 杜玮南 温进坤 韩梅.人血清对氧磷酶活性测定方法的建立及临床初步应用[J].河北医科大学学报,1999,20(3):134-136
11 邵军刘智广SDS-聚丙烯酰氨凝胶电泳检测尿激酶纯度[J]第四军医大学学报 1993,14(2):158-160
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