用终止剂改进超氧化物歧化酶邻苯三酚测活法

以二硫苏糖醇(DTT)或L-抗坏血酸(Vit C)为终止剂改进的经典邻苯三酚法测定超氧化物歧化酶(SOD)活性.反应体系:50mmol/L邻苯三酚,pH8.20,总体积9ml,25℃,加一滴DTT(100mmol/L)或Vit C(5%),约50μl,终止自氧化反应.终止后,反应体系的420nm光吸收在lh内保持恒定.终止剂法的邻苯三酚自氧化率及酶活测定灵敏度与经典法相近,一个酶活单位相当于纯SOD 100μg/L.     

棉铃虫核型多角体病毒sod基因在大肠杆菌中的表达

用PCR方法从棉铃虫(Helicoverpa armigera)单粒包埋型核型多角体病毒(HaSNPV) C1株基因组中扩增sod基因编码区,克隆到pGEM-T-easy vector,测定了核苷酸序列.将基因编码区克隆到原核表达载体pETblue2,构建了含表达质粒pETblue2/HaSNPV SOD,转化大肠杆菌DE3(BL21)进行IPTG诱导表达. SDS-PAGE分析表明SOD的表达量约为细胞总蛋白的37%.邻苯三酚法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物校正酶活力单位为694U/mg.     

棉铃虫核型多角体病毒sod基因在大肠杆菌中的表达

用PCR方法从棉铃虫(Helicoverpa armigera)单粒包埋型核型多角体病毒(HaSNPV) C1株基因组中扩增sod基因编码区,克隆到pGEM—T—easy vector,测定了核苷酸序列。将基因编码区克隆到原核表达载体pETblue2,构建了含表达质粒pETblue2／HaSNPV SOD,转化大肠杆菌DE3 (BL21)进行IPTG诱导表达。SDS—PAGE分析表明SOD的表达量约为细胞总蛋白的37％。邻苯三酚法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物校正酶活力单位为694U／mg。     

邻苯三酚自氧化—化学发光法测定SOD活性

有关超氧化物歧化酶(superoxide dis-mutase,SOD)活力测定方法已有不少研究。人们根据各自的条件建立起快速、有效的SOD测定分析手段。本文介绍一种以邻苯三酚为O_2~-发生系统的化学发光测定方法。并通过对测定条件的研究,得到测定的最佳方案,证明了方法的可行性和稳定性。     

莲子Fe-SOD的提取及其耐高温能力的初探

通过对莲子中超氧化物歧化酶(Super Oxide Dimutase,SOD)进行分离提纯,证实其是Fe-SOD,并观察Fe-SOD的耐热性,为SOD的提取和应用开辟了新的思路【。方法】以热变性与有机溶剂沉淀联合法分离提纯莲子Fe-SOD;利用Fe-SOD活性受H2O2的抑制而不受氰化物的抑制的特性来鉴定;设置不同的温度观察提取额莲子SOD的耐热性。【结果】莲子提取酶液活性为90.522U/ml,SOD酶液对邻苯三酚自氧化的抑制率为96.3%。莲子SOD活性受H2O2的抑制而不受氰化物的抑制。在75℃时保温30min后,酶活力仍然保留60%以上,在100℃保温30min时酶活力保留35%。【结论】以热变性与有机溶剂沉淀联合法分离提纯莲子SOD并鉴定其为耐热性Fe-SOD。     

一种小白菜叶细胞溶质铜锌超氧化物歧化酶的纯化和性质研究

<正> 超氧化物歧化酶(SOD)是生物体内重要的金属酶。有关植物SOD同工酶的研究文献报道较多,但对酶的纯化及性质研究报道较少。本文简要报道一种小白菜叶细胞溶质Cu·Zn-SOD的纯化及其性质。 材料为小白菜(Brassica chinensis L.),品种上海青。酶活力测定用本室改良的邻苯三酚自氧化法。蛋白质测定按文献[2]进行。酶纯化方法参照文献[3]。酶活性染色参照文     

一种SOD的测活方法——邻苯三酚自氧化法的改进

超氧化物歧化酶(SOD)是在医疗上具有广泛应用前景的酶。它的测活方法很多,一般是根据SOD具有抑制O_2介导的反应的能力这一原理而建立的。邻苯三酚自氧化法是其中较为简便的一种,本文对这种方法做了下述改进。材料与方法 1.仪器 M750UVIS-A型微量紫外可见分光光度计和日本岛津双光束双波长自动记录分光光度计。 2.试剂 SOD(上海生化所东风试剂厂产品),其它试剂均为分析纯。 3.邻苯三酚自氧化速率的测定取4.5ml100mM缓冲液(见表1),4.2ml蒸馏水,混匀     

橙皮苷对小鼠抗氧化作用及抗氧化酶基因表达的影响

目的探讨橙皮苷(HDN)对机体抗氧化作用的影响。方法采用分光光度法、邻苯三酚自氧化法和Fe2+-邻二氮菲法检测HDN体外清除自由基、抑制线粒体肿胀和红细胞氧化溶血;实验小鼠灌服不同浓度HDN(0、80、160、320 mg/kg)连续12 d,ELISA和分光光度法检测小鼠组织中MDA含量,抗氧化酶(SOD、CAT、GSH-PX)活力,RT-PCR技术分析抗氧化物酶mRNA表达水平。结果与对照组比较,HDN组自由基(·OH、O2-·、DPPH·)清除率明显提高,小鼠红细胞体外氧化溶血和线粒体肿胀显著降低;小鼠组织及血清中MDA含量降低,抗氧化酶SOD、CAT、GSH-PX活力明显高于对照组,组织中抗氧化酶mRNA(SOD、CAT、GSH-PX)表达上调。结论 HDN能够清除自由基,降低自由基引起的细胞氧化损伤,抑制过氧化物生成,上调抗氧化酶基因表达及提高酶活力,呈现良好的抗氧化作用。     

真菌菌丝发酵液抗氧化活性检测

目的检测长期保存的食用真菌发酵液中超氧化物歧化酶(SOD)活性和谷胱苷肽过氧化物酶(GSH-Px)活性。方法选取3株经实验室长期保存的食用真菌发酵液,采用邻苯三酚自氧化法测SOD酶活性;用二硫代二硝基苯甲酸(DTNB)直接法测GSH-Px酶活性。结果3种发酵液中SOD酶活性和GSH-Px酶活性均为阳性。结论食用菌发酵液中不仅测得SOD,还含有GSH-Px,而且经长期保存仍有很强的酶活性。     

磁处理酒对小鼠脑组织自由基代谢的影响

目的:探讨磁处理酒对小鼠脑组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)和胆固醇含量的影响。方法:用邻苯三酚测定SOD,TBA法测定MDA,改善的Griess法测定NO,高铁—醋酸—硫酸显色法测定胆固醇。结果:与对照组比较,磁酒组MDA含量显著提高(p<0.05);磁酒组NO含量与白酒组相比显著降低(p<0.05);白酒组NO含量明显高于对照组(p<0.05)。结论:磁处理白酒可影响小鼠脑组织的自由基代谢及胆固醇含量。     

Comparison of four indirect methods for fluid superoxide dismutase activities

Relatively small sample dilutions could render fluid extracellular (EC) superoxide dismutase (SOD) activity assays more subject to interfering compounds than tissue SOD assays. Highly variable relative SOD activity were obtained when comparing four indirect assays for several fluid samples (human plasma, human synovial fluid, and plasma from healthy or inflamed rats). Analysis of rat plasma fractionated with Sephadex G-150 showed that each assay (three xanthine oxidase based assays plus a modified pyrogallol assay) detected apparent SOD almost entirely at the same molecular weight as rat lung EC SOD. However, unfractionated fluid samples caused interferences with the xanthine oxidase based SOD assays, though not with the pyrogallol method. Examples of interference were stimulation of xanthine oxidase activity, color formation without xanthine oxidase, color formation despite excess Cu-Zn SOD addition, and absorbance changes with cyanide inhibition of EC SOD that were above or below blank values. In summary, relative fluid SOD values depended on the assay used, and a modified pyrogallol assay was not subject to several interferences found for three xanthine oxidase based assays of fluid SOD activity.     

Aluminum effect on the activity of superoxide dismutase and of other antioxygenic enzymes in vitro

The effect of Al on superoxide dismutase (SOD) and on other antioxygenic enzymes: horseradish peroxidase, catalase, and glutathione peroxidase, has been investigated in vitro. In the case of SOD, the effect of metal chelators (EDTA and deferoxamine) and a possible synergistic effect with iron salts have also been tested using the pyrogallol assay. There is no significant inhibitory effect of Al on the activity of any of the above-mentioned enzymes. Noticeable increases in SOD activity were observed when metal chelators were added to the medium, but not when high concentrations of Al were present too, in the case of deferoxamine (DFO). The former fact seems to be a consequence of the chelation of transition metal ions that catalyze pyrogallol autoxidation by a mechanism not inhibitable by SOD, interfering in its action, which may account for part of the DFO antioxidant effect observed in vivo. The latter phenomenon could be owing to a saturation of the chelating capacity of DFO by an excess of Al present in the medium, which should bring the system back to the interfering conditions explained above. It can be concluded that Al, either in the presence or in the absence of iron salts, does not inhibit SOD activity in vitro. Moreover, no significant binding of Al to SOD was demonstrated, and the amounts of its metal constituents, Cu and Zn, were not affected by preincubation of the enzyme with Al. The effect of the different compounds tested on the rate of autoxidation of the indicating scavenger, pyrogallol, and a suitable hypothesis on their role in the oxidation process are also discussed.     

Superoxide dismutase and glutathione peroxidase activities in erythrocytes as indices of oxygen loading in disease: a survey of one hundred cases

It has been established that the pyrogallol autoxidation method for the estimation of the activity of superoxide dismutase (SOD) (EC 1.15.1.1) is superior in precision and sensitivity to a superoxide-generating method (NADH/phenazine methosulfate linked to nitroblue tetrazolium reduction). Reference intervals were established in an urban population in the Far East for SOD activity in erythrocytes using the pyrogallol method, and for glutathione peroxidase (GSH-Px) (EC 1.11.1.9) activity in erythrocytes using a standard glutathione reductase-linked method. On this basis, erythrocyte SOD activities were significantly (P less than 0.05) depressed in cases of visceral cancer, acute myocardial infarct, congestive heart failure, respiratory failure, chronic renal failure, and diabetes mellitus, but within the reference interval in cases of lung cancer and asthma. Erythrocyte GSH-Px activity was significantly (P less than 0.05) depressed in cases of diabetes mellitus and chronic renal failure but elevated in respiratory failure and asthma. GSH-Px and SOD activities were well correlated in patients but not in the reference population.     

Species-specific heterogeneity for molecular weight estimates of serum extracellular superoxide dismutase activities.

1. Several apparent molecular weights (mol. wt) are reported for plasma or serum extracellular superoxide dismutase (EC SOD) activity. This study found species-dependent heterogeneity for apparent mol. wt using gel filtration with Sephadex G-150. 2. EC SOD activity in rabbit and guinea-pig serum, measured by a modified pyrogallol assay, eluted just before ceruloplasmin activity, but rat and bovine serum activity eluted after ceruloplasmin (apparent mol. wt of 142,000 and 73,000, respectively). 3. The heterogeneity between rat and rabbit serum was not eliminated by substituting a cytochrome-c-based SOD assay for the pyrogallol method, by substituting lung extracts for serum, by analysing a mixture of rat and rabbit serums, nor by analysing hemolysed serum. The apparent mol. wt of bovine serum EC SOD activity was not duplicated by gel filtration analysis of a mixture of bovine cytosolic SOD and albumin. 4. In conclusion, species-specific variation in apparent mol. wt for serum EC SOD activity was demonstrated under several circumstances.     

Important Roles of Minor Peroxide Components in the Autoxidation of Methyl Linoleate

Superoxide dismutase (SOD)-like compounds and activators of SOD were screened for in the extracts of fruits, vegetables, and mushrooms by measuring their effects on pyrogallol autoxidation, which is catalyzed by superoxide anion. SOD-like activity was high in aqueous extract of nameko, garlic, broccoli, and oriental lettuce. Ethanolic extracts of onion and watermelon could enhance human SOD activity more than 40%.     

Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt₂)₂ was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728 nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5 min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.     

Enhanced superoxide dismutase activity of pulsed cytochrome oxidase

The superoxide dismutase (SOD) activity of beef heart cytochrome oxidase, both in the resting (as isolated) and pulsed (reduced and reoxidized) states, has been investigated using their ability to inhibit the autoxidation rate of pyrogallol and epinephrine. Resting oxidase showed variable SOD activity, while in the pulsed state the SOD activity of cytochrome oxidase (CcO) increased by an order of magnitude. These results are discussed in terms of a physiological role for the pulsed oxidase.     

鸦肝超氧化物歧化酶的纯化

本文用低浓度氯化钠与肝脏一起匀浆,75℃加热,硫酸铵分级沉淀,在Cu~(2+)存在下透析,sephadex G-75柱层析等方法,从寒鸦肝脏中纯化出铜锌超氧化物歧化酶。对其理化性质鉴定表明,用此法纯化的SOD为均一性纯酶,比活性为4734U/mg pr,分子量32.6kD,紫外吸收峰在258.6nm。理化性质与文献报道的不同来源的同类酶基本相同。     

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification,biochemical characterization,and development of a plate microassay for activity

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.     

Oxidative stress contributes to vascular endothelial dysfunction in heart failure

Congestive heart failure (HF) is characterized by inadequate nitric oxide (NO) production in the vasculature. Because NO is degraded by oxygen radicals, we hypothesized that NO is degraded faster in HF from inadequate peripheral arterial antioxidant reserves. HF was induced in male Sprague-Dawley rats by left coronary artery ligation. Vascular endothelial function was evaluated by measuring the NO-mediated vasorelaxation response to acetylcholine (ACh; 10(-9)-10(-4) M) in excised aortas. This was repeated with the free radical generator pyrogallol (20 microM) and again with pyrogallol and superoxide dismutase (SOD; 60 U/ml). Aortic and myocardial SOD activity was also determined. ACh-induced vasorelaxation was reduced in HF (n = 9) compared with normal control rats (n = 11; P < 0.001). Pyrogallol further reduced vasorelaxation in HF: 74 +/- 11% at 10(-4) M ACh versus 58 +/- 10% in normal control rats (P < 0.004). There was a trend (P = 0.06) toward reduced SOD activity in HF aortas. In conclusion, altered NO-dependent vasorelaxation in HF is in part due to excessive degradation of NO and is likely related to reduced vascular SOD activity.