猪血超氧化物歧化酶的提取、性质及其化学修饰研究
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摘要
本课题是吉林省长春市科技局科技支撑计划项目(08KZ26)——“猪副产品深加工技术开发与产业化应用研究”的一部分。本研究以猪血为实验原料,主要对猪血中含有的超氧化物歧化酶(SOD)的分离提取及其性质进行了研究。
主要研究内容包括以下几个方面:首先,对猪血进行预处理,优化出最佳的热变性条件,对比分析了盐析法和有机溶剂沉淀法对提取超氧化物歧化酶的影响,并采用柱层析法对分离的超氧化物歧化酶进行了纯化。第二,采用高效液相色谱法(HPLC)和聚丙烯酰胺凝胶电泳法(SDS-PAGE)两种方法鉴定了猪血SOD的纯度,采用气相色谱法(GC)检测了纯品中丙酮溶剂的残留量。第三,在纯化的猪血SOD基础上,研究了SOD的主要酶学性质并初步分析了SOD的分子结构。第四,对猪血SOD进行了化学修饰,并对猪血SOD修饰产物进行了检测与分析。
Superoxide dismutase (SOD) is a kind of important antioxidant enzymes in the organism and it can remove the excessive oxygen free radicals in metabolic process. SODs are classified into four groups depending on their metal cofactor: Mn-SOD, Cu/Zn-SOD, Fe-SOD and Ni-SOD. Due to its antioxidative effects, SOD has been applied in protecting humans from radiation damage, aging and a wide range of common diseases, including cancer, cardiovascular, autoimmune, inflammatory diseases and some tumors.
The content of SOD in porcine blood is much higher than in other animal blood. In the present paper, an efficient and rapid isolation and purification process for porcine blood SOD is proposed and maked full use of waste blood resources in slaughtering industry. The purity of the porcine blood SOD was studyed on the nature and chemical modification, to make the enzyme more reasonably applicated to medical, industry and chemistry field, etc.
This paper developed five main kinds of work. The conclusions are listed as follow:
1. The results of the separation and purcification of the SOD from porcine blood showed that:
(1) From the single factor experiment and three dimensions orthogonal polynomial regressive experimental design, it is found that three dimensions orthogonal polynomial regressive equation of the thermal denaturation method is ^y_(比活力) =-4.862+5.252Z_1+0.568Z_2+43.235Z_3 (where Z_1:warm-up time/min, Z_2:warm-up temperature /℃,Z_3: the copper sulfate concentration /%); The regressive analysis tells us that multiple correlation coefficient R=0.889 and multiplicities determination coefficient R~2=0.790. The optimum conditions of thermal denaturation method are: warm-up time: 20min;warm-up temperature: 55℃;the copper sulfate concentration:3.5%,the enzyme activity was 337.49U/ml and the enzyme specific activity was 280.77 U/mg.
(2) Salt fractionation and organic solvent precipitation method are compared, and it showed that organic solvent precipitation method is better. The enzyme specific activity was 2077.83 U/mg by organic solvent precipitation method. When equal volume of cold acetone was added into the enzyme extraction, the enzyme activity and the enzyme specific activity were all reached the highest.
(3) The elution profile of the Sephadex G-75 chromatography of purified SOD is showed that the enzyme activity is 3965 U/ ml and the enzyme specific activity was 9675.60 U/mg.
2. The analysis on the purity of the porcine blood SOD and determination of the acetone residue showed that:
(1) Measured by high performance liquid chromatography, the retention time of porcine blood superoxide dismutase was 16.2min and the purity was 80.3%.
(2) Analyzed by discontinuous polyacrylamide gel electrophoresis, in the electrophoresis gels showed a single protein band, and the protein staining and activity staining band corresponding with each other showed that have reached the electrophoretically pure enzyme.
(3) Detected by gas chromatography system, the acetone residue of the purified SOD was 0.21%.The acetone residue was completely consistent with organic solvent residual requirement in pharmacopoeia of the People's Republic of China (2005 edition) 2 AppendixⅧP.
3. From the analysis of nature of SOD from the porcine blood, it can be found that:
(1) The porcine blood SOD contains copper and zincum, so the enzyme in purified porcine blood was indicated as Cu/Zn-SOD.
(2) Porcine blood SOD was detected by the ultraviolet absorption spectra, and the results indicated that the maximum absorbance is at 262.5nm. The fluorescence spectrum of porcine blood SOD showed the maximum excitation wavelength at 294nm and the maximum emission wavelength at 336nm.
(3) The purified enzyme activity showed good thermostability for 1h at 50℃and 60℃.
The enzyme retained more than 50% of its activity after incubation in the buffer at pH between 6 and 8 for 1h. The results showed that porcine superoxide dismutase also had good pH value stability.
(4) The SDS-PAGE electrophoresis of purified porcine blood SOD suggested its homogeneous quality. The rough size of the subunit determined by SDS-PAGE was 16218Da.Cu/Zn-SOD consists of two identical subunits, thus it can calculate molecular weight of its holoenzyme 32436Da.
(5) The atomic absorption spectrophotometer (AAS) was used to determine the Cu contents in the samples. And the results indicated that the Cu contents in purified porcine blood SOD was 0.223mg/g. Besides, the verification test of AAS was conducted and the results showed that the RSD of precision was 1.840%, the RSD of reproducibility was 0.975% and both of the RSD were all less than 2%, which showed that the AAS was reliable to analyze the Cu contents in the samples. At the same time it identified that the porcine superoxide dismutase was Cu/Zn-SOD again.
(6) This experiment was adopted to study on SOD oxidation resistance by researching of the extent of anti-browning of fruit. The results showed that, with the extension of time, apple surface oxidation and dried out wrinkled degree of daubing after SOD powder was relatively lighter than not daubing SOD powder apple. This showed that superoxide dismutase played the antioxidant role.
4. It could be found when study on the optimum process conditions of modifying porcine blood SOD and SOD stability after modification that:
(1) We have studied the modification of superoxide dismutase using the activation of polyethylene glycol, taking the modified SOD activity remaining and modified rate as parameter, investigating the effects of the modified quality ratio, modification temperature, the modified time and pH value on the modified rate and enzyme activity, achieving the optimum conditions for modifying superoxide dismutase. The results showed that the best conditions are: the best mass ratio of SOD and activation PEG for 1.3:3, the modification temperature for 4℃, the modification time for 90min and the modification pH value for 8.
(2) This experiment was modified superoxide dismutase by the optimum process conditions and collected superoxide dismutase with 1.6×50 cm column of a Sephadex G-75. The results showed that the enzyme activity was declined and the biggest enzyme activity was 2533U/ml.This may be that enzyme side chain residues or activity center of amino acid was modified, thus affecting the enzyme activity.
(3) Through studying on SOD stability after modification, it showed that heat resistance, enzyme resistant, acid and alkali resistant capabilities of the modified SOD are stronger than natural SOD. Detected the modified superoxide dismutase by the ultraviolet absorption spectra, it gave the maximum absorbance at 263nm. The results showed that before and after the modification, enzyme ultraviolet absorption spectral characteristic was consistent.
主要研究内容包括以下几个方面:首先,对猪血进行预处理,优化出最佳的热变性条件,对比分析了盐析法和有机溶剂沉淀法对提取超氧化物歧化酶的影响,并采用柱层析法对分离的超氧化物歧化酶进行了纯化。第二,采用高效液相色谱法(HPLC)和聚丙烯酰胺凝胶电泳法(SDS-PAGE)两种方法鉴定了猪血SOD的纯度,采用气相色谱法(GC)检测了纯品中丙酮溶剂的残留量。第三,在纯化的猪血SOD基础上,研究了SOD的主要酶学性质并初步分析了SOD的分子结构。第四,对猪血SOD进行了化学修饰,并对猪血SOD修饰产物进行了检测与分析。
Superoxide dismutase (SOD) is a kind of important antioxidant enzymes in the organism and it can remove the excessive oxygen free radicals in metabolic process. SODs are classified into four groups depending on their metal cofactor: Mn-SOD, Cu/Zn-SOD, Fe-SOD and Ni-SOD. Due to its antioxidative effects, SOD has been applied in protecting humans from radiation damage, aging and a wide range of common diseases, including cancer, cardiovascular, autoimmune, inflammatory diseases and some tumors.
The content of SOD in porcine blood is much higher than in other animal blood. In the present paper, an efficient and rapid isolation and purification process for porcine blood SOD is proposed and maked full use of waste blood resources in slaughtering industry. The purity of the porcine blood SOD was studyed on the nature and chemical modification, to make the enzyme more reasonably applicated to medical, industry and chemistry field, etc.
This paper developed five main kinds of work. The conclusions are listed as follow:
1. The results of the separation and purcification of the SOD from porcine blood showed that:
(1) From the single factor experiment and three dimensions orthogonal polynomial regressive experimental design, it is found that three dimensions orthogonal polynomial regressive equation of the thermal denaturation method is ^y_(比活力) =-4.862+5.252Z_1+0.568Z_2+43.235Z_3 (where Z_1:warm-up time/min, Z_2:warm-up temperature /℃,Z_3: the copper sulfate concentration /%); The regressive analysis tells us that multiple correlation coefficient R=0.889 and multiplicities determination coefficient R~2=0.790. The optimum conditions of thermal denaturation method are: warm-up time: 20min;warm-up temperature: 55℃;the copper sulfate concentration:3.5%,the enzyme activity was 337.49U/ml and the enzyme specific activity was 280.77 U/mg.
(2) Salt fractionation and organic solvent precipitation method are compared, and it showed that organic solvent precipitation method is better. The enzyme specific activity was 2077.83 U/mg by organic solvent precipitation method. When equal volume of cold acetone was added into the enzyme extraction, the enzyme activity and the enzyme specific activity were all reached the highest.
(3) The elution profile of the Sephadex G-75 chromatography of purified SOD is showed that the enzyme activity is 3965 U/ ml and the enzyme specific activity was 9675.60 U/mg.
2. The analysis on the purity of the porcine blood SOD and determination of the acetone residue showed that:
(1) Measured by high performance liquid chromatography, the retention time of porcine blood superoxide dismutase was 16.2min and the purity was 80.3%.
(2) Analyzed by discontinuous polyacrylamide gel electrophoresis, in the electrophoresis gels showed a single protein band, and the protein staining and activity staining band corresponding with each other showed that have reached the electrophoretically pure enzyme.
(3) Detected by gas chromatography system, the acetone residue of the purified SOD was 0.21%.The acetone residue was completely consistent with organic solvent residual requirement in pharmacopoeia of the People's Republic of China (2005 edition) 2 AppendixⅧP.
3. From the analysis of nature of SOD from the porcine blood, it can be found that:
(1) The porcine blood SOD contains copper and zincum, so the enzyme in purified porcine blood was indicated as Cu/Zn-SOD.
(2) Porcine blood SOD was detected by the ultraviolet absorption spectra, and the results indicated that the maximum absorbance is at 262.5nm. The fluorescence spectrum of porcine blood SOD showed the maximum excitation wavelength at 294nm and the maximum emission wavelength at 336nm.
(3) The purified enzyme activity showed good thermostability for 1h at 50℃and 60℃.
The enzyme retained more than 50% of its activity after incubation in the buffer at pH between 6 and 8 for 1h. The results showed that porcine superoxide dismutase also had good pH value stability.
(4) The SDS-PAGE electrophoresis of purified porcine blood SOD suggested its homogeneous quality. The rough size of the subunit determined by SDS-PAGE was 16218Da.Cu/Zn-SOD consists of two identical subunits, thus it can calculate molecular weight of its holoenzyme 32436Da.
(5) The atomic absorption spectrophotometer (AAS) was used to determine the Cu contents in the samples. And the results indicated that the Cu contents in purified porcine blood SOD was 0.223mg/g. Besides, the verification test of AAS was conducted and the results showed that the RSD of precision was 1.840%, the RSD of reproducibility was 0.975% and both of the RSD were all less than 2%, which showed that the AAS was reliable to analyze the Cu contents in the samples. At the same time it identified that the porcine superoxide dismutase was Cu/Zn-SOD again.
(6) This experiment was adopted to study on SOD oxidation resistance by researching of the extent of anti-browning of fruit. The results showed that, with the extension of time, apple surface oxidation and dried out wrinkled degree of daubing after SOD powder was relatively lighter than not daubing SOD powder apple. This showed that superoxide dismutase played the antioxidant role.
4. It could be found when study on the optimum process conditions of modifying porcine blood SOD and SOD stability after modification that:
(1) We have studied the modification of superoxide dismutase using the activation of polyethylene glycol, taking the modified SOD activity remaining and modified rate as parameter, investigating the effects of the modified quality ratio, modification temperature, the modified time and pH value on the modified rate and enzyme activity, achieving the optimum conditions for modifying superoxide dismutase. The results showed that the best conditions are: the best mass ratio of SOD and activation PEG for 1.3:3, the modification temperature for 4℃, the modification time for 90min and the modification pH value for 8.
(2) This experiment was modified superoxide dismutase by the optimum process conditions and collected superoxide dismutase with 1.6×50 cm column of a Sephadex G-75. The results showed that the enzyme activity was declined and the biggest enzyme activity was 2533U/ml.This may be that enzyme side chain residues or activity center of amino acid was modified, thus affecting the enzyme activity.
(3) Through studying on SOD stability after modification, it showed that heat resistance, enzyme resistant, acid and alkali resistant capabilities of the modified SOD are stronger than natural SOD. Detected the modified superoxide dismutase by the ultraviolet absorption spectra, it gave the maximum absorbance at 263nm. The results showed that before and after the modification, enzyme ultraviolet absorption spectral characteristic was consistent.
引文
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