反相胶束体系对辣根过氧化物酶结构与功能的影响

在十六烷基三甲基溴化铵（ＣＴＡＢ）／异辛烷－正戊醇反相胶束中,研究了含水量（Ｗ０）和表面活性剂对辣根过氧化物酶（ＨＲＰ）和活力的影响机制。在测定不同含水量（Ｗ０）和ＣＴＡＢ不同浓度下的ＵＶ－Ｖｉｓ光谱（即Ｓｏｒｅｔ吸收光谱）及活力的变化的基础上,发现含水量不同时,反相胶束主要通过影响ＨＲＰ的活性中心而影响酶的活力,但ＣＴＡＢ对酶活性中心没有明显影响。此外通过反相胶束与水相中的ＨＲＰ与Ｈ２Ｏ２复合物     

反胶束萃取血红蛋白的研究

研究了ＣＴＡＢ－正辛醇－正庚烷交束溶液萃取牛血红蛋白（ｐＨｂ）时、ｐＨ值、表面活性剂浓度、助表面活性剂浓度、离子种类和离子强度、溶剂比以及蛋白质浓度等因素对萃取效果的影响,并以蛋白质分子与表面活性剂分子间的相互作用以及反胶束大空间阻碍作用上进行了解释。研究表明,水相ＰＨ值在１０．５￣１２．５之间,ＫＣ１浓度为０．１ｍｏｌ／ｌ,反胶束溶液中表面活性剂浓度为０．０２ｍｏｌ／ｌ,正辛醇与正庚烷之比为０．     

高产稳产聚羟基烷酸的重组大肠杆菌的构建

重组大肠杆菌Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＨＭＳ１７４（ｐＴＺ１８ＵＰＨＢ） 含有携带聚羟基烷酸（ＰＨＡ） 合成基因（ ｐｈａＣＡＢ）＊＊ 的质粒ｐＴＺ１８ＵＰＨＢ,是很有潜力的ＰＨＡ 生产菌,但存在着质粒不稳定和不能合成３羟基丁酸（３ＨＢ） 与３羟基戊酸（３ＨＶ） 共聚物［Ｐ（３ＨＢｃｏ３ＨＶ）］ 的缺陷。将ＲＫ２ 质粒上的ｐａｒ ＤＥ 基因引入ｐＴＺ１８ＵＰＨＢ 构成质粒ｐＪＭＣ２ ,该质粒可以在宿主Ｅ．ＣｏｌｉＨＭＳ１７４ 中稳定遗传。将培养基中的磷酸盐浓度降至１８ ｍ ｍｏｌ／Ｌ,发现Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） 能够以丙酸为前体合成Ｐ（３ＨＢｃｏ３ＨＶ） ,其中３ＨＶ 在共聚物中的含量为５ ％ ～８ ％ 。在５Ｌ自动发酵罐中分批补料培养Ｅ．Ｃｏｌｉ ＨＭＳ１７４（ｐＪＭＣ２） ,培养基初始磷酸盐浓度为１５ ｍ ｍｏｌ／Ｌ,３０ ｈ 后每升培养液中干菌体可达４２－５ ｇ,Ｐ（３ＨＢｃｏ３ＨＶ） 占干重的７０ ％ ,其中３ＨＶ 在共聚物中的含量为４－９ ％ 。     

氧化修饰高密度脂蛋白对培养人动脉平滑肌细胞细胞周期蛋白D_1基因表达的影响

动脉平滑肌细胞（ｓｍ ｏｏｔｈ ｍ ｕｓｃｌｅ ｃｅｌｌ,ＳＭＣ）是动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）斑块中的主要细胞,它的增殖在ＡＳ形成过程中极其重要．利用体外培养的人主动脉ＳＭＣ,观察了天然高密度脂蛋白（ｎａｔｉｖｅ ｈｉｇｈ ｄｅｎｓｉｔｙ ｌｉｐｏｐｒｏｔｅｉｎ,Ｎ－ＨＤＬ）及氧化修饰ＨＤＬ（ｏｘｉｄｉｚｅｄ ＨＤＬ,ＯＸ－ＨＤＬ）对培养人主动脉ＳＭＣ ｃｙｃｌｉｎ Ｄ１（细胞周期蛋白Ｄ１）基因转录表达的影响．结果表明：（１）Ｎ－ＨＤＬ对ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达无影响（Ｐ＞ ０．０５）;（２）ＯＸ－ＨＤＬ使ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达显著增强（Ｐ＜０．０１）,其表达量随时间（２、１２、２４ ｈ）延长而增加．上述结果表明,ＯＸ－ＨＤＬ的致ＡＳ作用可能与其刺激ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达增加有关．     

脂肪酶在反相胶囊中的催化行为的研究

系统研究了脂肪酶在ＡＯＴ／水／异辛烷反应相胶囊中的催化行为。在一定条件下,反相胶囊中的酶反应的仍符合Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学原理,研究了含水量,底物浓度,ｐＨ,温度,溶剂的种类和表面活性剂浓度等对酶反应的影响。结果表明,酶活力与Ｒ值（水与表面活性剂的摩尔比值）有关。获得最大酶活力的条件是Ｒ＝１１,ｐＨ７．０,温度３２．５℃,橄榄油浓度为４０％。     

水稻叶片中过氧化氢与核酮糖—1,5—二磷酸羧化酶／加氧酶降解 …

甲基紫精（ＭＶ）处理水稻植株能快速诱导核酮糖－１,５－二磷酸羧化酶／加氧酶（Ｒｒｂｉｓｃｏ,ＥＣ４．１．１．３９）及其它可溶性蛋白的降解。ＭＶ浓度越高,降解速率越高。ＭＶ能诱发叶片内源Ｈ２Ｏ２迅速积累。光合电子传递抑制剂ＤＣＭＵ（１５０μｍｏｌ／Ｌ）能显著抑制ＭＶ（１００μｍｏｌ／Ｌ）诱导的Ｒｕｂｉｓｃｏ及其它可溶性蛋白的降解;活性氧清除剂抗坏血酸（５ｍｍｏｌ／Ｌ）、甘露醇（１０ｍｍｏｌ／Ｌ）、苯     

细胞转化过程中胞内钙离子浓度的变化

使用钙离子荧光探针Ｆｌｕｏ－３ＡＭ和ＤＮＡ荧光染料Ｈｏｅｃｈｓｔ３３３４２联染细胞的方法,利用显微分光光度计ＭＰＶⅡ测量了两种温度敏感细胞—６Ｍ２和ｔｓＲＳＶＮＩＨ３Ｔ３－ＬＡ９０细胞及Ｃ３Ｈ１０Ｔ１／２和转化Ｃ３Ｈ１０Ｔ１／２细胞在不同细胞周期时相内钙的浓度,发现从Ｇ１期到Ｓ期到Ｇ２期,６ｍ２细胞当在３３℃培养时（转化状态）胞内钙的浓度〔Ｃａ＾２＋〕ｉ分别为８５．０,１３８．４２３９．０ｎｍｏｌ     

天然及氧化修饰脂蛋白对人动脉平滑肌细胞原癌基因表达的影响

动脉平滑肌细胞（ＳＭＣ）的增殖在动脉粥样硬化（ＡＳ）的形成过程中极其重要。我们在建立人主动脉ＳＭＣ体外培养方法的基础上,观察了ＬＤＬ,ＶＬＤＬ及ＨＤＬ和相应的氧化修饰型脂蛋白对培养人ＳＭＣｓｉｓ,ｊｕｎ,Ｈ－ｒａｓ原癌基因及Ｒｂ抗癌基因转录表达的影响。结果表明：（１）ＨＤＬ对ＳＭＣｓｉｓ,ｊｕｎ,ｒａｓ基因表达无影响;（２）ＬＤＬ和ＶＬＤＬ有使这些基因表达增加的趋势;（３）ｏｘ－ＬＤＬ,ｏｘ－ＶＬＤＬ和ｏｘ－ＨＤＬ具有使ＳＭＣｓｉｓ,ｊｕｎ,和ｒａｓ基因表达显著增强的作用（Ｐ＜０．０１）,且其作用较相应的天然脂蛋白大（Ｐ＜０．０１）;（４）天然和氧化修饰型脂蛋白对Ｒｂ基因表达均无影响。据上述结果推测：ＬＤＬ,ＶＬＤＬ,ｏｘ－ＬＤＬ,ｏｘ－ＶＬＤＬ和ｏｘ－ＨＤＬ的致ＡＳ作用可能与刺激ＳＭＣｓｉｓ,ｊｕｎ和ｒａｓ原癌基因表达增加有关。     

变色马兜铃中银袋醌的分子结构研究

从变色马兜铃Ａｒｉｓｔｏｌｏｓｈｉａ ｖｅｒｓｉｃｏｌａｒ Ｓ．Ｍ．Ｈｗａｎｇ的根中分离到一个新的倍半萜醌,命名为银袋醌（Ｖｅｒｓｉｃｏｑｕｉｎｏｎｅ）,橙黄色短毛发状结晶,ｍｐ２９０－２９６℃（分解）,分子式Ｃ１５Ｈ１６Ｏ５,依靠１Ｈ－１３Ｃ ＮＭＲ ＣＯＬＯＣ谱和选择性远程ＤＥＰＴ谱,其分子结构补阐明为４,８－二羟基－３,６－二甲基－５－（２－羟基异丙基）－１,２－萘二酮。体外试验对人肝癌细胞     

磷化氢、二氧化碳混合气体对腐食酪螨成螨的生物学效应

本文研究结果表明：腐食酪螨Ｔｙｒｏｐｈａｇｕｓｐｕｔｒｅｓｃｅｎｔｉａｅ（Ｓｃｈｒａｎｋ）成螨在０％、８％、１６％（容积比）ＣＯ２气体中耗氧量随ＣＯ２浓度的增加而增加,当在３２％、６４％ＣＯ２气体中,该成螨的耗氧量反倒低于其在正常大气中的耗氧量。在０％、８％、１６％、３２％、６４％ＣＯ２与０．０５ｍｇ／ＬＰＨ３混合气体中该成螨对ＰＨ３的吸收量分别为１．１１±０．９２、１．７９±０．５６、５．１４±１．１３、７．６０±１．８０、８．０８±０．８５μｇ／ｈｒ’ｇ,在同一ＣＯ２浓度条件下试螨对ＰＨ３的吸收量在高浓度ＰＨ３（０．４５ｍｇ／Ｌ）中明显大于在低浓度ＰＨ３（０．０５ｍｇ／Ｌ）中,但ＰＨ３吸收量的增加倍数远远低于ＰＨ３浓度的增加倍数。ＰＨ３对该螨过氧化氢酶的抑制体内酶高于离体酶,细胞色素ｃ氧化酶受ＰＨ３抑制则相反。被ＰＨ３抑制的过氧化氢酶和细胞色素Ｃ氧化酶活性恢复时间分别为二周和一周。本文还对ＰＨ３的可能杀螨机理及ＣＯ２在此过程中的作用进行了讨论     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

The role of the extent of hydration of reversed micelles of surfactant s in the regulation of the peroxidase activity of ferritin and immunocomplexes of cortisol-peroxidase conjugates

The effect of the degree of hydration (W0) of reversed micelles of Aerosol OT (AOT) and its mixture with Triton X-45 in heptane on the peroxidase activity of horse spleen ferritin in the oxidation of various substrates by hydrogen peroxide and organic hydroperoxides and on the activity of solubilized or immobilized immunocomplexes of horseradish peroxidase-cortisol conjugates (HP-COR) was studied. The peroxidase activity versus W0 plot has maxima at W0 8-14 and 19-22, which cannot be attributed to dissociation of immunocomplexes into its components or of ferritin into its subunits. The possibility of the stabilization of the conformers of oligomeric proteins by reversed micelles and the effect of the self-association of micelles on the peroxidase activity of the HP-COR immunocomplexes and ferritin were discussed. A procedure for the isolation of the iron-containing cluster from the ferritin molecule without reduction of the Fe3+ ions was suggested.     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

A methylene blue-mediated enzyme electrode for the determination of trace mercury(II), mercury(I), methylmercury, and mercury-glutathione complex

A methylene blue-mediated enzyme biosensor has been developed for the detection of inhibitors including mercury(II), mercury(I), methylmercury, and mercury-glutathione complex. The inhibition to horseradish peroxidase was apparently reversible and noncompetitive in the presence of HgCl2 in less than 8 s and irreversibly inactivated when incubated with different concentrations of HgCl2 for 1-8 min. The binding site of horseradish peroxidase with HgCl2 probably was a cysteine residue SH. Mercury compounds can be assayed amperometrically with the detection limits 0.1 ng ml(-1) Hg for HgCl2 and methylmercury, 0.2 ng ml(-1) Hg for Hg2(NO3)2 and 1.7 ng ml(-1) Hg for mercury glutathione complex. Inactivation of the immobilized horseradish peroxidase was displayed in the AFM images of the enzyme membranes.     

Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Vanadium effect on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase in vitro.

The effect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase has been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 microM). No significant inhibitory effects were found when V(V) was tested with catalase and when either V(IV) or V(V) were assayed with glutathione peroxidase. For the latter, the effect of V on the different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent inhibitory dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with the metal. Besides, no significant binding of V(IV) or V(V) to the enzyme could be demonstrated. The lack of a direct inhibitory effect of V on the activity of the main antioxidant enzymes suggests that many biological and toxicological effects of V may be mediated more by oxidative reactions of the metal or of its complexes with physiologically relevant biomolecules than by a direct modulation of enzymatic activities.     

The phenomenon of super activity in dihydrofolate reductase entrapped inside reverse micelles in apolar solvents

Bovine liver dihydrofolate reductase has been solubilized in reverse micelles of cationic surfactant cetyltrimethylammonium bromide (CTAB) in isooctane-chloroform (1:1,V/V) mixture. Variation of waterpool (WO), pH and surfactant concentration showed that the enzyme activity was regulated by these parameters and was higher than the activity found in aqueous buffer (defined as superactivity); the maximum being at WO 13.3, pH 7.0 and CTAB concentration 75 mM. The Michaelis constants, Km for the substrate FAH2 and NADPH were found to be greater than those determined in water. Since reverse micelles have some features similar to those of biomembranes, display of super activity by dihydrofolate reductase indicates that enzymes in vivo may possess higher activity than actually observed in vitro studies in aqueous solutions.     

Anomalous oxidation of MDL 73,404 by horseradish peroxidase

3,4-Dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium-4-methylbenzene sulfonate (MDL 73,404) is a cardioselective water-soluble quaternary ammonium analogue of Vitamin E which is synthesized to augment the antioxidant defence in situations of free radical injury such as myocardial infarction/reperfusion. Its oxidation by any peroxidative enzyme has not been studied kinetically. This paper describes its enzymatic oxidation by horseradish peroxidase (HRP). The activity was followed spectrophotometrically at 255nm, and the experimental results were simulated using the program "KINETIC 3.1" for Windows 3.x. The MDL 73,404 was oxidized by horseradish peroxidase in the presence of H2O2 to its corresponding MDL 73,404 quinone. During this oxidation, the horseradish peroxidase showed an unexpectedly slow kinetic response with time, which contrast with the linear product accumulation curve measured with 2,2'-azino-bis-(3-estilbenzotiazol-6-sulfonic acid) (ABTS). This response was dependent on the respective concentrations of enzyme, MDL 73,404 and H2O2. However, when the enzyme was incubated with H2O2, the slow kinetic response disappeared and a lag period was observed. Furthermore, when p-coumaric acid (PCA) was added, the activity increased and the slow kinetic response became a straight line. In order to explain this anomalous behaviour, a kinetic model has been proposed and its differential equations simulated. From the correlation between experimental and simulated results it is concluded that MDL 73,404 can act as a slow response substrate for peroxidase, probably due to the presence of a quaternary ammonium side chain that confers on it a slow capacity to convert compound III into ferriperoxidase.     

Enhanced dye decolorization efficiency by citraconic anhydride-modified horseradish peroxidase

Bromophenol blue and methyl orange removal capabilities of citraconic anhydride-modified horseradish peroxidase were compared with those of native horseradish peroxidase. Citraconic anhydride-modified horseradish peroxidase showed higher decolorization efficiencies for both dyes than native horseradish peroxidase. Upon the chemical modification, the decolorization efficiencies were increased by 1.8% and 12.4% for bromophenol blue and methyl orange, respectively. The quantitative relationships between decolorization efficiencies of dyes and reaction conditions were also investigated. Experimental data revealed that aqueous phase pH, reaction time, temperature, enzyme concentration and ratio of dye and H₂O₂ play a significant role on the dye degradation. Lower dose of citraconic anhydride-modified horseradish peroxidase was required than that of native enzyme for the decolorizations of both dyes to obtain the same decolorization efficiencies. Citraconic anhydride-modified HRP exhibited a good decolorization of dye over a wide range of dye concentration from 8 to 24 or 32 μmol l⁻¹ at 300 μmol l⁻¹ H₂O₂, which would match industrial expectations. Kinetic constants for two different dyes were also determined. Citraconic anhydride-modified horseradish peroxidase shows greater affinity and catalytic efficiency than native horseradish peroxidase for both dyes.