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1.
Alpha-methylacyl-CoA racemase (AMACR) is over-expressed in many cancer types and can serve as a novel diagnostic biomarker. Development of convenient and sensitive detection methods of AMACR is of particular importance for cancer diagnosis. Aptamers are a type of recognition elements, which possess many advantages over antibody, making them suitable for applications in biosensing and biotechnology. In this work, we use the efficient surface modification of gold nanoparticles (AuNPs) to prepare the horseradish peroxidase (HRP) and aptamer dual-functionalized nanoprobe. The immobilization of HRP and thiol-terminated aptamer on the surface of AuNPs can be achieved through electrostatic interaction and the formation of Au–S bond, respectively. This nanoprobe, which is used as discriminating and catalytic probe, can be combined with enzyme immunoassay method to increase the detection sensitivity of AMACR. The detection limit can reach as low as 4.6 pg mL−1 due to the dual signal amplification from enzymatic cycling and the high loading of enzymes on AuNPs. This sensitivity is about three orders of magnitude higher than that of AMACR aptamer based fluorescence method, which is also comparable to or one order of magnitude higher than that of ELISA. Furthermore, this method is more simple and effective, which not only avoids the conjugation between recognition element and the catalytic enzyme, but also achieves greater signal amplification. This assay could be used as a sensitive and selective platform for the detection of target protein. 相似文献
2.
The possibility to afford by a biomimetic reaction ‘unnatural’ products, which could offer a better bioactivity profile than natural analogues, is outlined and the first applications to the synthesis of lignans and related compounds have been reported. Here we describe the synthesis of new heterodimers, having a phenylcoumaran skeleton, by horseradish peroxidase catalyzed cross-coupling reactions of methyl esters of substituted hydroxycinnamic acids. 相似文献
3.
Palligarnai T. Vasudevan Luting Olivia Li 《Applied biochemistry and biotechnology》1996,60(3):203-215
Studies of the kinetic behavior of horseradish peroxidase (HRP) at pH 8 and at room temperature indicate that the reaction
of phenol with H2O2 catalyzed by HRP exhibits normal Michaelis-Menten saturation kinetics. An irreversible reaction mechanism for the steady-state
kinetics of HRP, which is consistent with the experimental data, is considered. The second-order rate constants for the reactions
of HRP with H2O2 and compound II with phenol are 4.14 × 105 M-1s-1 and 5.54 × 104M-1s-1, respectively. 相似文献
4.
《Journal of separation science》2003,26(18):1701-1706
Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators. 相似文献
5.
Horseradish peroxidase (HRP) catalyzes the oxidation of a wide variety of compounds inthe presence of hydroperoxide. The products have been used as the important materialsin extensive fields because of the con juged bond. Many investigators have stUdied themechanism of HRP. When native HRP reacts with hydroperoxide, compound i, which istwo oxidizing equivalents of the ferric state of HRP, is formed. Compound I issubsequently reduced by one electron to form compound 11. Reduction of compo… 相似文献
6.
辣根过氧化物酶对心肌线粒体过氧化损伤的保护作用 总被引:1,自引:0,他引:1
以黄嘌呤氧化酶/次黄嘌呤自由基诱生系统建立了稳定的离体心肌线粒体的损伤模型,并应用此模型研究辣根过氧化物酶对心肌线粒体自由基氧化损伤的保护作用,结果表明,辣根过氧化物酶能抑制心肌线粒体中氧自由基的形成,降低膜脂的过氧水平,保护了线粒体膜酶活性,有效地防止自由基对线粒体结构和功能的损伤。 相似文献
7.
通过采用邻苯二甲酸酐(PA)对辣根过氧化物酶(HRP)的蛋白链进行修饰, 研究了PA化学修饰对HRP的稳定性、催化活性、活性中心结构、酶对底物的亲合性和专一性等催化性质的影响. 结果显示: 酸性条件下(pH=3), 4小时后PA-HRP的催化活性比天然HRP提高了7.5%;碱性条件下(pH=10), 4小时后PA-HRP的催化活性比天然HRP提高了27%. PA-HRP的K_m值为8.16 (mmol/L), 小于天然HRP的K_m值12.99 (mmol/L), 而PA-HRP的k_(cat)/K_m值为7.86(10~4(L/ mmol· min)大于天然HRP的k_(cat)/K_m的6.70(10~4(L/ mmol· min). 这些催化活性和动力学数据表明了PA-HRP与天然HRP相比其稳定性、酶对底物的亲和性和专一性得到了提高. 紫外-可见光谱、拉曼光谱数据显示: 修饰剂PA 改变了天然HRP血红素周围的微环境, 对酶蛋白分子的活性中心结构并没有影响. 差示光谱显示PA修饰剂可以提高酶对底物的亲和力. 相似文献
8.
9.
利用对辣根过氧化物酶催化体系的干扰测定左旋多巴 总被引:3,自引:2,他引:3
提出辣根过氧化物酶法分析左旋多巴,方法基于一定条件下左旋多巴对辣根过氧化物酶催化对氯苯酚-4-AAP偶联发色体系有干扰,干扰程度与左旋多巴的有浓度有相关性。 相似文献
10.
11.
酚类聚合物在水相胶束中的酶促合成 总被引:3,自引:1,他引:3
本文报道辣根过氧化物酶在水相胶束中催化聚合取代酚类的研究。水相胶束中酶催化活力高,产物分子量均一;通过调节表面活笥剂和单体的浓度,可以有效地控制产物的分子量,从而建立了不同聚合度有机纳米材料酶促合成的新方法。产物具有较强的蓝紫色荧光及较好的耐热性能,是一种很有应用前景的蓝光材料。 相似文献
12.
Leirião PR Fonseca LJ Taipa MA Cabral JM Mateus M 《Applied biochemistry and biotechnology》2003,110(1):1-10
A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 μmol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads
were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a
batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support
(55.8U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention
of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 μmol of H2O2 reduced/(h·Uimmob HRP). 相似文献
13.
An organic-phase enzyme electrode (OPEE) based on horseradish peroxidase (HRP) immobilized within Nafion on spectroscopic graphite was investigated in acetonitrile. The amperometric electrode response to hydrogen peroxide and cumene hydroperoxide present was found to be the result of the reduction of oxygen, produced upon enzymatic decomposition of both hydroperoxides (i.e., by the catalase-like activity of HRP). The electrode response was found to depend linearly on the hydroperoxide concentration up to 700 M within the range of potentials from –200 to –400 mV (versus Ag|AgCl). Detection limits of approximately 45 M for H2O2 and 100 M for cumene hydroperoxide were determined under the selected experimental conditions. Nernstian dependence (the open circuit voltage of HRP-based electrode versus logarithm of H2O2 concentration) was obtained between 0.2 and 2.0 mM, with a slope of approximately 23 mV per logarithmic unit, suggesting a catalase-like, two-electron disproportionation of the substrate in acetonitrile. 相似文献
14.
Differences between the rates of oxidation of substrates by horseradish peroxidase in buffer, aqueous micellar solution of
Brij 35 and in reverse micelles formed of Brij 30 in several hydrocarbons are discussed.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
反相胶束对辣根过氧化物酶催化反应的影响 总被引:3,自引:0,他引:3
胶束体系是酶学研究比较理想的体系,因为它所具有的诸如热力学稳定、光学透明及能增溶亲水分子、亲油分子或两性分子等性质,使许多酶在胶束体系中的反应速率远远高于在水相中,即人们发现的所谓“超活性”[‘j.辣根过氧化物酶(HRP)是一种比较稳定的酶,且价廉易得,具备一般过氧化物酶的典型反应.在研究中人们发现,HRP在反相胶束体系中同样具有“超活性”,由于HRP能够催化大量底物进行反应,因此“超活性”对HRP的催化反应具有重要意义.已有研究者[’、’j对CTAB反相胶束体系中HRP的性质进行了探讨,但反相胶束对HRP的… 相似文献
16.
17.
研究了辣根过氧化物酶(HRP)/H2O2体系中苯酚与4-氨基安替比林(4-AAP)的偶合反应。在pH 8.0的条件下,产物在494.0 nm处有最大吸收,线性范围为(4.3×10-6~2.6×10-4)mol/L(R2=0.9993),检出限为4.75×10-7mol/L,相对标准偏差为3.7%(n=9)。动力学方面,用孤立法研究了各反应物对反应速率的影响,得到4-AAP、H2O2和phenol的反应级数分别为0、1.3和0.7,据此建立了反应速率方程式,求得活化能为42.09 kJ/mol,并推测出反应的可能机理。 相似文献
18.
The synthesis of nitroanilines catalyzed by horseradish peroxidase(HRP) in the presence of sodium nitrite and hydrogen peroxide was investigated, o-Nitroaniline and p-nitroaniline were found in the nitrated products. 2-Aminotoluene and 4-aminotoluene could also be nitrated to give corresponding nitrated products. This protocol has great potentials to open new avenues useful for the synthesis of nitroaniline and its derivatives. 相似文献
19.
辣根过氧化物酶在表面活性剂膜中的直接电化学 总被引:14,自引:0,他引:14
利用3种表面活性剂分别将辣根过氧化氢酶固定在裂解石墨棱面(edge-plane pyrolytic graphite,EPG)电极表面,研究了辣根过氧化物酶(HRP)中Fe(Ⅲ)/Fe(Ⅱ)电对与电极之间的直接电子传递过程以及酶催化双氧化还原过程。实验结果表明:(1)表面活性剂是一种固定酶的理想材料;(2)这种体系可能构造第三代生物传感器,对解释生物体代谢过程具有理论意义,对制备第三代生物传感器具有应用价值。 相似文献
20.
Antonin Tuynman Hans E. Schoemaker Ron Wever 《Monatshefte für Chemie / Chemical Monthly》2000,131(6):687-695
Summary. Horseradish peroxidase (HRP), myeloperoxidase (MPO), and manganese peroxidase (MnP) have been shown to catalyze the asymmetric
sulfoxidation of thioanisole. When H2O2 was added stepwise to MPO, a maximal yield of 78% was obtained at pH 5 (ee 23%), whereas an optimum in the enantiomeric excess (32%, (R)-sulfoxide) was found at pH 6 (60% yield). For MnP a yield of 18% and a high enantiomeric excess of 91% of the (S)-sulfoxide were obtained at pH 5 and a yield of 36% and an ee of 87% at pH 7.0. Optimization of the conversion catalyzed by horseradish peroxidase at pH 7.0 by controlled continuous addition of hydrogen peroxide during turnover and monitoring the presence of native enzyme as
well as of intermediates I, II, and III led to the formation of the sulfoxide in high yield (100%) and moderate enantioselectivity
(60%, (S)-sulfoxide).
Received November 18, 1999. Accepted January 21, 2000 相似文献