Investigation of the effects of natural compounds isolated from Arum rupicola var. rupicola on glutathione reductase enzyme purified from bovine liver

Glutathione reductase (GR, E.C. 1.8.1.7), a flavoenzyme, is responsible for recycling of oxidized glutathione disulfide. This study was performed in two main sections. In the first GR was purified from bovine liver by affinity column chromatography and the purification rate and specific activity of the enzyme were calculated as 1832‐fold and 141 EU/mg protein, respectively. The subunit molecular weight of the enzyme was determined as 55 kDa by means of SDS–PAGE. The second section isolated natural components of Arum rupicola Boiss. var. rupicola using column chromatography. The isolation protocol for this plant was performed with a series of different‐sized columns with hexane–ethyl acetate. According to the thin‐layer chromatography plate, seven substances (R1–R7) were isolated. Our study's aim was to find new activators or inhibitors for GR activity. With this aim, all isolated substances were tested for GR activity. R6 showed competitive inhibition, while R4 had noncompetitive inhibition of GR activity. R1 played a role as an activator of GR activity. The inhibitory activity percentage vs. concentration graph was plotted. Values of IC₅₀ for R4 and R6 were calculated as 0.193 mg/mL and 3.98 μg/mL, respectively, from the equation of this graph.     

Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.     

Biosensing approach for glutathione detection using glutathione reductase and sulfhydryl oxidase bienzymatic system

Chitosan membrane with glutathione reductase and sulfhydryl oxidase (SOX) was subsequently integrated onto the surface of spectrographic graphite rods for obtaining a glutathione biosensor. The working principle was based on the monitoring of O₂ consumption that correlates the concentration of glutathione during the enzymatic reaction. A linear relationship between sensor response and concentration was obtained between 0.5 and 2.0 mM for oxidized glutathione (GSSG), and 0.2–1.0 mM for reduced glutathione (GSH) in the presence of 2 μM nicotinamide adenine dinucleotide phosphate (NADPH) under the optimum working conditions. Also, reduced/oxidized glutathione were separated by HPLC and utility of bienzymatic system was investigated as an electrochemical detector for the analysis of these compounds. All data were given as a comparison of two systems: biosensor and diode array detector (DAD).     

Modeling,stability and the activity assessment of glutathione reductase from Streptococcus Thermophilus; Insights from the in-silico simulation study

Antioxidant enzymes (AEs) are the main parts of the natural barriers of the body which deactivate the oxidant factors. To discover and understand their structures and function will deserve a deeper investigation. Accordingly, as an AE of probiotic strains, glutathione reductase of Streptococcus thermophilus (GRst), is characterized and modeled by in-silico methods. The investigation indicated the physicochemical properties of the enzyme and estimated its half-life of being more than 10 h. The analysis revealed that the enzyme is composed of 86 strands, 123 helices, and 241 random coils. Homology modeling of the GRst led to the construction of the enzyme’s 3D model that 62% of which is analogous to the glutathione reductase of Escherichia Coli (GRec), and which is qualitatively high in terms of Molpdf, ERRAT, Verify-3D and Ramachandran scores. Moreover, the structural stability of the model was substantiated within 10 and 20 ns at 400 and 300 K, respectively. Interestingly, these data showed that the enzyme is more stable than GRec at 400 K. In other words, the active cavity of the constructed model is characteristic of 38 amino acid residues within 4 Å around the NADPH and GSSG as corresponding ligands of GRst. Noteworthy, herein is the fact that, CYS40 and CYS45 are specified as the active site residues of this enzyme. Furthermore, the interaction assays of the model support its antioxidant capability which is even more than that of GRec.In general, these data provide a new model of AEs being inclusive of high antioxidant capacity and thermostability.     

两步柱色谱法分离纯化重组肿瘤血管生长抑制因子Kringle 5

建立了一种用Ni2+螯合的Chelating Sepharose Fast Flow亲和柱色谱和Sephadex G-75凝胶排阻柱色谱分离纯化重组肿瘤血管生长抑制因子Kringle 5的方法。采用该工艺得到的重组Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明其纯度约为98%,且具有抑制鸡胚绒毛尿囊膜新生血管生成的生物活性。     

Fluorescence detection of glutathione reductase activity based on deoxyribonucleic acid-templated silver nanoclusters

Fluorescent silver nanoclusters stabilized by DNA (DNA-AgNCs) exhibit distinct response rates to thiol and disulfide. Glutathione reductase can catalyze the reduction of the oxidized glutathione (GSSG) quickly to reduced glutathione (GSH) in the presence of β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH). Consequently, DNA-AgNCs can serve as a new fluorescent platform for assaying the glutathione reductase (GR) activity. This newly proposed assay has a high sensitivity and a good selectivity toward GR. The GR activity can be detected in the range of 0.2–2.0 mU mL⁻¹ with a minimum detectable concentration of 0.2 mU mL⁻¹. Pepsin, lysozyme, trypsin, avidin, thrombin, myoglobin, and BSA have little effect on the fluorescence intensity of DNA-AgNCs. The GR activity assay is successfully used to monitor the inhibition of GR activity by a typical inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea.     

Purification of porcine reproductive and respiratory syndrome virus from cell culture using ultrafiltration and heparin affinity chromatography

Porcine reproductive and respiratory syndrome (PRRS) virus is the causative agent of the most significant infectious disease currently affecting the swine industry worldwide. Density gradient ultracentrifugation remains the most commonly used method for porcine reproductive and respiratory syndrome virus (PRRSV) purification. However, this technique has notable drawbacks including long processing time and limited processing volume in each run. To overcome these limitations, a scalable process was developed. PRRSV propagated in MARC-145 was released by three freeze/thaw cycles. After a low speed centrifugation step, the virus particles in the supernatant were concentrated twice by an ultrafiltration step. The ultrafiltration step concentrated the virions effectively with no detectable loss while some cultural/cellular proteins were removed. The virions in the ultrafiltration retentate were then applied to a heparin affinity column on a fast performance liquid chromatography unit. The combined ultrafiltration and heparin affinity chromatography process removed more than 96% of cellular and medium proteins. During a stepwise elution strategy, the viral particles were eluted at two separate peaks recovering 27.5% and 25.4% of viral particles loaded onto the column with a purity of 194 and 3917 particles/μg protein, respectively.     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

微囊藻毒素-RR的纯化及鉴定

将固相萃取和凝胶色谱技术相结合,建立了以野外采集的水华蓝藻为原料提取和纯化微囊藻毒素-RR(MC-RR)的有效方法。用70%的甲醇溶液溶解35 g藻浆,经离心等系列处理,得粗提液,旋转蒸发去除甲醇;粗提液经HLB柱固相萃取后,得到7.5 mL洗脱液,然后将其浓缩至2 mL,再用Sephadex LH-20凝胶色谱柱分离纯化,洗脱液分管收集,用紫外分光光度计测定每管洗脱液在238 nm的吸收值,并绘制洗脱曲线。使用高效液相色谱对峰值组分进行鉴定,同时利用紫外分光光度计对毒素的光谱特征进行鉴定。最终获得3.65 mg纯度超过90%的MC-RR样品,产品得率为74.1%。其紫外吸收光谱在238 nm处有特征吸收,证明所纯化的样品为MC-RR。     

灵芝发酵液中蛋白酶抑制剂GLPIA2的纯化及其特性

采用乙醇分级沉淀、凝胶色谱纯化、阴离子交换色谱分离等步骤从灵芝深层发酵液中提取得到蛋白酶抑制剂GLPIA1 与GLPIA2。其中GLPIA2仅在215 nm处有紫外吸收,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定为单一条带,相对分子质 量为15000。由其氨基酸组成分析谱图可看出,其酸性氨基酸含量较高,碱性氨基酸及芳香族氨基酸含量较低。GLPIA2抑 制剂的底物特异性研究表明,它对天冬氨酸族的胃蛋白酶和酵母蛋白酶A有相对较强的抑制作用。     

高效液相色谱-电感耦合等离子体质谱联用同时测定鸡肉与鸡肝中的7种砷形态

建立了一种同时测定阿散酸、亚砷酸根、砷酸根、砷胆碱、砷甜菜碱、一甲基砷和二甲基砷等7种砷形态的高效液相色谱-电感耦合等离子体质谱分析方法。样品采用人工胃液作为提取液进行超声处理,再以碳酸铵溶液和水作为流动相,采用阴离子分析柱将样品提取液进行分离,最后进入电感耦合等离子体质谱测定。各砷形态在1～50 μg/kg范围内线性关系良好,相关系数r2均在0.999以上;在1、2、10 μg/kg 3个添加水平进行了方法学验证,平均回收率为84.3%～106.6%,相对标准偏差为1.4%～4.2%; 7种砷形态的定量限均为1 μg/kg。方法重现性好、灵敏度高、前处理简单,适用于鸡肉和鸡肝中主要有机砷和无机砷残留的分析检测。     

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

Purification of recombinant proteins by chemical removal of the affinity tag

The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.     

离子交换色谱法分离纯化鸡卵黄免疫球蛋白

建立了高效、经济、大规模获得鸡卵黄免疫球蛋白(IgY)的生产方法。在对传统的水稀释法改良的基础上,结合聚乙二醇沉淀与离子交换色谱进行IgY的分离纯化。结果显示,用8倍无菌水稀释蛋黄液,用0.1 mol/L HCl调节pH为5.2,在4 ℃下静置8 h,于5000×g力离心可得上清粗IgY液,经测定回收率可达93.47%。然后用6%聚乙二醇沉淀后经DEAE-Toyopearl 650 M离子交换纯化,最佳的纯化条件: 0.05 mol/L磷酸盐缓冲液(PBS, pH 7)平衡上样,0.075 mol/L PBS(pH 7)洗脱。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果显示所得的IgY的纯度为95.02%,活性保持率高达73.77%。本研究弥补了传统分离方法不能同时达到高纯度和高回收率的缺点,且可用于大规模生产。     

Purification of neutral protease by dye affinity chromatography

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme^™, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25°C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8°C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25°C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.     

一种从蛇毒中纯化神经生长因子的新工艺

为了能从中华眼镜蛇蛇毒中简单快速地分离纯化神经生长因子（一种治疗各种神经性损伤和神经退行疾病的药物,简称NGF）,采用不同的色谱柱联用的方式对NGF的纯化工艺进行了研究。结果表明,采用DEAE Sepharose F.F.和Sephadex G 50二步柱色谱工艺可以从蛇毒中快速分离得到神经生长因子。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳和反相高效液相色谱分析, 证明所得到的NGF为单一组分,相对分子质量约为 29 000。对8　d龄鸡胚背根神经节采用体外培养法,结果证明,所得NGF具有明显的促进神经纤维     

The production and properties of a new xylose reductase from fungus

Neurospora crassa XI was found to ferment xylose and glucose simultaneously. Xylose was the appropriate inducer for the production of xylose reductase that had two isoenzymes designated as EI and EII. Both EI and EII, which were purified by affinity chromatography, had NADPH-dependent xylose reductase activities. EII also had NADH-dependent activity, and EI is the only xylose reductase found so far without any NADH-dependent activity. EI and EII had MWs of 30 kDa and 27 kDa, and pIs of 5.6 and 5.2, respectively. The specifities of EI and EII against triose, pentoses, and hexoses were studied. The K_ms against xylose for EI and EII were 2.3 mM and 1.1 mM respectively, which were much lower than those of the xylose reductase from yeast.     

1,3-Oxazole derivatives of cytisine as potential inhibitors of glutathione reductase of Candida spp.: QSAR modeling,docking analysis and experimental study of new anti-Candida agents

Natural products as well as their derivatives play a significant role in the discovery of new biologically active compounds in the different areas of our life especially in the field of medicine. The synthesis of compounds produced from natural products including cytisine is one approach for the wider use of natural substances in the development of new drugs. QSAR modeling was used to predict and select of biologically active cytisine-containing 1,3-oxazoles. The eleven most promising compounds were identified, synthesized and tested. The activity of the synthesized compounds was evaluated using the disc diffusion method against C. albicans M 885 (ATCC 10,231) strain and clinical fluconazole-resistant Candida krusei strain. Molecular docking of the most active compounds as potential inhibitors of the Candida spp. glutathione reductase was performed using the AutoDock Vina. The built classification models demonstrated good stability, robustness and predictive power. The eleven cytisine-containing 1,3-oxazoles were synthesized and their activity against Candida spp. was evaluated. Compounds 10, 11 as potential inhibitors of the Candida spp. glutathione reductase demonstrated the high activity against C. albicans M 885 (ATCC 10,231) strain and clinical fluconazole-resistant Candida krusei strain. The studied compounds 10, 11 present the interesting scaffold for further investigation as potential inhibitors of the Candida spp. glutathione reductase with the promising antifungal properties. The developed models are publicly available online at http://ochem.eu/article/120720 and could be used by scientists for design of new more effective drugs.     

IDA型Cu~(2 )-螯合亲和膜色谱法对牛肝过氧化氢酶纯化的研究(英文)

首次采用以改性纤维素为基质、亚氨二乙酸（ＩＤＡ）为取代配基的铜离子螯合膜色谱法对牛肝过氧化氢酶（ＢＬＣ）的分离纯化进行了研究。缓冲液的ｐＨ值对ＢＬＣ与螯合配基的结合影响显著。在选定的色谱条件下，ＢＬＣ粗酶液经ＩＤＡ型Ｃｕ２＋－螯合膜色谱柱一步纯化，比活性平均提高４．７倍，回收率为６７．７％。金属螯合膜色谱柱可用含０．２ｍｏｌ／Ｌ的咪唑或５０ｍｍｏｌ／ＬＥＤＴＡ－１ｍｏｌ／ＬＮａＣｌ的缓冲液再生，反复使用，后者比前者对柱子的再生效果更好。