铜离子对烟草多酚氧化酶变性和复性影响的热力学研究(英)

在有无5 mmol·L^-1 CuSO₄存在的两种情况下，运用荧光光谱研究了烟草多酚氧化酶在盐酸胍诱导下的变性和复性平衡。烟草多酚氧化酶在6.0 mol·L^-1盐酸胍变性30 min(25 ℃)即完全失活。荧光光谱结果表明：铜离子能够提高烟草多酚氧化酶的结构稳定性和抗盐酸胍变性的能力，进而影响烟草多酚氧化酶在盐酸胍诱导下的变性和复性过程。没有外源铜存在的情况下，盐酸胍诱导的烟草多酚氧化酶变性和复性是一个可逆的二态过程；在5 mmol·L^-1 CuSO₄存在的条件下，由于结合了Cu²⁺的酶的中间态稳定性增加，显示特征荧光，结果显示，外源铜存在时，盐酸胍诱导的烟草多酚氧化酶变性和复性是一个可逆的三态过程。根据相应模型，进行了热力学计算。上述实验结果得到酶活性测定的进一步证实，在6 mol·L^-1盐酸胍中放置5，10，15，20，25，30 min，烟草多酚氧化酶的剩余活性分别为18.7%，11.7%，8.9%，6.6%，3.6%，0.06%，但在5 mmol·L^-1 CuSO₄存在的条件下，剩余活性则分别为60.3%，44.6%，42.5%，40.2%，25.6%，25.3%。     

硫酸铜诱导烟草多酚氧化酶抗变性的研究

In order to investigate the interaction between exogenous Cu2+ and Polyphenoloxidase (PPO) from Nictiana Tobacum, the Cu2+-induced resistant-denaturation against guanidine hydrochloride(Gdn-HCl) was studied by the following enzymatic activity assay, fluorescence spectroscopy and circular dichroism (CD). The results show that the GdnHCl-induced unfolding is a two-state process with no detectable inter mediate state in the absence of exogenous Cu2+, while the GdnHCl-induced unfolding in the presence of 10.0 mmol/L exogenous Cu2+ follows a three-state transition with an intermediate state, which results from the fact that Cu2+ increases the structural stability of native PPO and its intermediate. In terms of enzymatic activities, 6 mol/L GdnHCl makes PPO lose 81.4% of its original activity after 5 min, inactivate completely after 30 min, while in the presence of 10.0 mmol/L exogenous Cu2+, only 39.4% and 75.1%, after 5 and 30 min respectively. According to the CD measurements, the relative average fractions of α-helix, anti-parallel2-sheet, 2-turn/parallel 2-sheet, aromatic residues and disulfide bond, and random coil/γ-turn are 1.1%, 3.8%, 3.3%, 7.5% and 84.3%, respectively, in the 6.0mol/L GdnHCl containing no CuSO4, but34.2%, 13.7%, 21.0%, 9.5% and 21.6%, respectively, in the same concentration of Gdn-HCl containing 10mmol/L CuSO4.     

曲安奈德的荧光光谱法研究

提出了测定曲安奈德 (TA)的简单、灵敏的荧光光谱新方法。实验条件包括浓硫酸、加热温度及时间、溶剂、β CD的用量及CTMAB的影响 ,建立了两种荧光光谱法测定TA的新体系 :(1)CTMAB体系 ,线性范围为 0～ 4 .6× 10 -6mol/L ,检出限为 3.5 9× 10 -8mol/L ;(2 ) β CD与乙醇体系 ,线性范围为 0～ 2 .3× 10 -6mol/L ,检出限为 1.9× 10 -8mol/L。用于实际药物试样分析 ,获得满意结果。     

脲或盐酸胍溶液中变性溶菌酶复性过程中集聚体的研究

建立在蛋白质变性-复性三态模型的基础上, 给出了一个描述在变性液中变性蛋白质复性时蛋白质浓度和其复性率的关系式. 通过这个关系式, 可以获得两个重要的描述蛋白质变性-复性体系特征的参数, 一个是包含在一个集聚体分子中的变性蛋白质的分子数目n, 另一个是蛋白质从原始态到形成集聚体过程中的表观集聚平衡常数K. 以三种溶菌酶在脲和盐酸胍溶液中的变性-复性过程对此方程进行了验证, 结果表明所给出的方程能够很好地描述三种溶菌酶在这两种变性液中的复性结果, 三种溶菌酶在两种变性液中有形成二分子集聚体的趋势. 变性溶菌酶在复性过程中的电泳和高效凝胶排阻色谱也同时能够监测到复性过程中集聚体的形成, 并且监测结果与上述方程所得的结果一致.     

吲哚美辛的荧光光谱分析

系统研究了荧光光谱法测定吲哚美辛的条件,包括酸度、溶剂、β-环糊精的用量及β-环糊精与溴化十六烷基三甲基铵(CTMAB)协同作用的影响,发现在pH10.8的缓冲溶液中,CTMAB和β-CD能增敏荧光,从而建立了测定吲哚美辛的高灵敏的荧光光谱分析方法,体系的线性范围为0～2.79×10-6mol／L,检出限为5.73×10-9mol／L;应用本法对市售吲哚美辛片剂进行测定,结果令人满意。     

碘化物-CTMAB体系共振光散射法测定电镀废水中的钯

用简易荧光计研究了在微酸性介质中钯-碘化钾-溴化十六烷基三甲基铵体系的共振光散射光谱,考查了光谱特征、影响因素和适宜的反应条件,确定了散射光强度与溶液中钯浓度的关系,提出了共振光散射法测定钯的方法。钯的线性范围为0.0~0.5μg/mL,检出限为0.005μg/mL。该法可用于电镀废水中钯浓度的测定。     

荧光法测定有机磷农药残留总量及其分析应用

在pH 6.20的Britton-Robinson缓冲溶液中,4,5-二溴荧光素(R)与一定浓度的溴化十六烷基三甲基铵(CTMAB)反应,使其荧光强度剧增,当在R-CTMAB体系中加入有机磷农药后,体系的荧光强度明显降低,且降低程度与有机磷农药的加入量呈良好的线性关系,据此建立了测定有机磷农药残留总量的新方法.在优化实验条件下,线性范围为0.05～0.88 mg/L,检出限为0.041 mg/L.已用于大米、面粉和土壤中有机磷农药残留总量的检测,回收率在87.5%～95.7%之间,相对标准偏差为1.5%～2.3%,符合农药残留量分析的要求.     

含盐酸胍β-乳球蛋白A冷、热变性的DSC法研究

运用差示扫描量热法研究了牛β-乳球蛋白A(β-Lg A)在含盐酸肌(GuHCl)的溶液中的冷、热变性过程.实验表明:在pH3以下,热变性后β-Lg A分子结合的质子数几乎不变;GuHCl的存在降低热变性过程活化能.在含2.50及3.06mol/LGuHCl的溶液中,观察到β-LgA既可冷变性又可热变性.冷变性及其复性过程是可再现的.一般地说,热变性过程不可再现.和热变性比较,冷变性过程除变性焓有相反符号外,它的活化能也更低,而且冷变性后伸展的多肽链结合的GuHCl分子数增加得更多.冷变性焓的绝对值大于热变性焓值,表明与盐酸肌的结合是放热性的,是氢键性质的.在热变性过程中单体分子间的协同性较强.用Ooi模型分析,按重原子数平均来说.β-LgA单体分子构象变化对总变性焓的贡献在球蛋白中最低.     

硝酸钠-硫氰酸铵-溴化十六烷基三甲基铵体系浮选铜

浮选分离;硝酸钠-硫氰酸铵-溴化十六烷基三甲基铵体系浮选铜     

双波长分光光度法测定溴化十六烷基三甲基铵

以二甲酚橙为显色剂,采用双波长分光光度法测定水中溴化十六烷基三甲基铵。实验确定的测定条件为:最大吸收波长为590 nm,等吸光度点的波长点为509 nm,选择pH为6.86的Na3PO4-Na2HPO4缓冲溶液3.50 mL,0.80 g/L二甲酚橙显色剂2.0 mL,显色时间为5 min。试剂加入顺序对测定结果基本无影响。CTMAB的质量浓度在0~1.2 mg/mL范围内符合比尔定律,标准曲线为ΔA=0.0123ρ 0.0825,相关系数为R2=0.9998,样品加标回收率为99.6%~101.3%。     

Characterization and Potential Applications of Immobilized Glucose Oxidase and Polyphenol Oxidase

Pyrrole functionalized polystyrene (PStPy) was copolymerized with pyrrole to obtain a conducting copolymer, P(PStPy‐co‐Py) which is used as the immobilization matrix. Glucose oxidase and polyphenol oxidase enzymes were immobilized via the entrapment method by electrochemical polymerization. Enzyme electrodes were prepared by electrolysis at a constant potential using sodium dodecyl sulfate (SDS) as the supporting electrolyte during the copolymerization of PStPy with pyrrole. Maximum reaction rates (V_max) and enzyme affinities (Michaelis‐Menten constants, K_m) were determined for the enzyme entrapped both in polypyrrole (PPy) and P(PStPy‐co‐Py) matrices. Optimizations of enzyme electrodes were done by examining the effects of temperature and pH on enzymes' activities along with the shelf life and operational stability investigations. Glucose oxidase enzyme electrodes were used for human serum analysis and glucose determination in two brands of orange juices. Polyphenol oxidase enzyme electrodes were used for the determination of phenolics in red wines of Turkey.     

Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant K_m was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn²⁺ and K⁺ could enhance the activity of PPO; that Ba²⁺ and Ca²⁺ could inhibit the activity of PPO; and that Cu²⁺ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.     

伏安式多酚氧化酶电极的研究

本报道了一种能性能良好的以吩嗪甲酯（ＰＭＳ）修饰的玻碳电极为基体电极的优安式多酚氧化酶电极。采用从蘑菇中粗提取的多酚氧化酶固定於电极表面，可用循环伏安法测定５×１０＾－＾７ｍｏｌ／Ｌ的多巴胺。本首次采用丙烯酸丁酯－丙烯酸甲酯共聚乳液固定酶，所制得电极性能与戊二醛交联法所得电极基本相同。在降低电极充电电流方面，采用半微分技术使电极性能得到很大改善，测定灵敏度达到２．５×１０＾－＾７ｍｏｌ／Ｌ，且     

Insights to Sequence Information of Polyphenol Oxidase Enzyme from Different Source Organisms

Polyphenol oxidases (PPOs) are widely distributed enzymes among animals, plants, bacteria, and fungi. PPOs often have significant role in many biologically essential functions including pigmentation, sclerotization, primary immune response, and host defense mechanisms. In the present study, forty-seven full-length amino acid sequences of PPO from bacteria, fungi, and plants were collected and subjected to multiple sequence alignment (MSA), domain identification, and phylogenetic tree construction. MSA revealed that six histidine, two phenylalanine, two arginine, and two aspartic acid residues were highly conserved in all the analyzed species, while a single cysteine residue was conserved in all the plant and fungal PPOs. Two major sequence clusters were constructed by phylogenetic analysis. One cluster was of the plant origin, whereas the other one was of the fungal and bacterial origin. Motif GGGMMGDVPTANDPIFWLHHCNVDRLWAVWQ was found in all the species of bacterial and fungus sources. In addition, seven new motifs which were unique for their group were also identified.     

Defense-Related Polyphenol Oxidase from Hevea brasiliensis Cell Suspension: Purification and Characterization

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70?kDa on sodium dodecyl sulfate?Cpolyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V _max /K _m ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH?6.0. The PPO activity was retained at pH?4.0?C10.0 and temperature 10?C60?°C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and ??-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.     

Cu2+与烟草多酚氧化酶相互作用研究

本文通过酶活性测定,荧光光谱和紫外光谱研究了外加Cu2+与烟草多酚氧化酶(简称PPO)的相互作用。结果表明,微量铜的加入能增加酶的活性,犤Cu2+犦/犤PPO犦为0.20左右时酶活性最大,犤Cu2+犦/犤PPO犦为0.91时,Cu2+开始表现出对PPO活性的抑制;Cu2+对PPO内源荧光的猝灭机制属于形成络合物所引起的静态猝灭,猝灭常数Ksv为8.0375×103L·mol-1;Cu2+的加入使PPO蛋白质分子构象发生变化,α-螺旋含量增加,多肽链及Trp和Tyr残基的芳杂环进一步向分子内收缩,疏水基团之间的疏水作用增强。     

莲藕多酚氧化酶与底物或抑制剂相互作用的光谱学研究

本文研究了纯化的莲藕多酚氧化酶(PPO)与底物和抑制剂相互作用时的二级结构变化。园二色谱分析表明莲藕PPO主要含有α-螺旋和β-折叠结构。与抑制剂作用后,莲藕PPO活性显著降低,同时伴随其二级结构中α-螺旋结构明显减少,表明莲藕PPO的活性中心可能位于α-螺旋结构中。荧光分析表明,莲藕PPO与邻苯三酚(pyrogallic acid,PA)作用后,酪氨酸残基荧光强度略有降低,λmax位移不明显,色氨酸残基荧光强度略有降低,λmax红移2 nm;而与莲藕多酚(Lotus root polyphenol,LRP)作用后,莲藕PPO分子中酪氨酸(Tyr)和色氨酸(Trp)残基荧光强度显著提高,且其最大发射波长分别蓝移6nm和红移5nm。当加入异Vc钠后,Tyr和Trp残基最大发射峰显著红移,说明Trp和Tyr残基位于一定的疏水环境对维持PPO催化活性的优势构象至关重要。     

儿茶酚在预活化聚酰胺膜多酚氧化酶电流传感器上的响应特性

本采用预活化聚酰膜固定多酚氧化酶并与浸蜡石墨极组合成传感器，测定儿茶酚的线范围２．×１０－７－１．２５×１０＾－５ｍｏｌ／Ｌ。检出限１．５×１０＾－７ｍｏｌ／Ｌ。酶的固定时间仅需１－２ｍｉｎ，５个月之后被固定酶的活性未见下降，活性酶膜的制备，贮存，替换均很方便，对传感器的机理进行了探讨。     

多酚氧化酶电化学发光生物传感器的研制及其应用

通过超声诱导法提取土豆浸取液中的多酚氧化酶.收集上层清液与纯石墨粉混合,经阴干并与液态石蜡研磨均匀后,将此混合物装入空心碳电极中,由此制成多酚氧化酶电化学发光生物传感器.以此传感器对肾上腺素进行电化学发光检测,肾上腺素在多酚氧化酶存在下被溶液中的溶解氧所氧化而产生过氧化氢,而所产生的过氧化氢与鲁米诺反应产生化学发光,此乃生化传感器的作用机理.由于肾上腺素的存在而使化学发光强度的增加与其质量浓度在2×10-4～3×10-3g·L-1范围内呈线性关系;该方法的检出限为6.0×10-5g·L-1.对4.0×10-4g·L-1肾上腺素分别测定8次,所得结果的相对标准偏差小于5%.用此方法分析了肾上腺素注射液样品,测得结果与标准值相符.在此注射液的基础上作加标回收试验,测得回收率在94%～99%之间.     

Polyphenol Oxidase as a Promising Alternative Therapeutic Agent for Cancer Therapy

Cancers have always been the most difficult to fight, the treatment of cancer is still not considered. Thus, exploring new anticancer drugs is still imminent. Traditional Chinese medicine has played an important role in the treatment of cancer. Polyphenol oxidase (PPO) extracted from Edible mushroom has many related reports on its characteristics, but its role in cancer treatment is still unclear. This study aims to investigate the effects of PPO extracted from Edible mushroom on the proliferation, migration, invasion, and apoptosis of cancer cells in vitro and explore the therapeutic effects of PPO on tumors in vivo. A cell counting kit-8 (CCK8) assay was used to detect the effect of PPO on the proliferation of cancer cells. The effect of PPO on cancer cell migration ability was detected by scratch test. The effect of PPO on the invasion ability of cancer cells was detected by a transwell assay. The effect of PPO on the apoptosis of cancer cells was detected by flow cytometry. Female BALB/c mice (18–25 g, 6–8 weeks) were used for in vivo experiments. The experiments were divided into control group, model group, low-dose group (25 mg/kg), and high-dose group (50 mg/kg). In vitro, PPO extracted from Edible mushroom significantly inhibited the proliferation, migration, and invasion capability of breast cancer cell 4T1, lung cancer cell A549, and prostate cancer cell C4-2, and significantly promoted the apoptosis of 4T1, A549, and C4-2. In vivo experiments showed PPO inhibitory effect on tumor growth. Collectively, the edible fungus extract PPO could play an effective role in treating various cancers, and it may potentially be a promising agent for treating cancers.