Spectrofluorimetric发展和动态验证的定量测定过氧化物酶在Pico摩尔量:应用天然植物组织的检测活动

Nelligere Arkeswaraiah Chamaraja 1 Padmarajaiah Nagaraja2, Honnur Krishna3

研究学者、研究化学,迈索尔大学Manasagangotri,迈索尔,卡纳塔克邦,India1
学系教授,研究化学,迈索尔大学Manasagangotri,迈索尔,卡纳塔克邦,India2
研究学者、研究化学,迈索尔大学Manasagangotri,迈索尔,卡纳塔克邦,India3

文摘

一个高度敏感的催化spectrofluorimetric过氧化氢和过氧化物酶的测定方法。这是基于para-acetylaminophenol氧化催化过氧化物酶的影响,非荧光化合物荧光探针,过氧化氢稍微基础培养基和反应机理的研究。随后的反应是spectrofluorimetrically通过测量荧光强度的21 - dihydroxy-5-51 diacetyldiaminebiphenyl(λex = 325海里,λem = 439海里)的固定时间5分钟的起始反应。在最优条件下,过氧化物酶可以确定在19 - 378点,s.d = 0.44的10次测量。方法的检出限降至0.6995点和定量限的值是2.33点。米歇利斯- Menten常数我¨我©m K和Vmax反应被发现103μM和1000分别最低为1。动力学参数如催化力量我¨过氧化氢过氧化氢我©马克斯m V K和催化效率我¨eff K = 1 /斜率[E] 0我©被发现是9.7087×106分别M-1min-1和2.3620×109 m - 1最低为1。方法的适用性测试样品一些蔬菜植物的过氧化物酶活性

关键字

荧光,催化,催化效率,蔬菜样本

介绍

过氧化物酶(提到过1.11.1.7)无处不在的酶催化各种拆分传氧反应和过氧化氢(H2O2)作为底物[1]。过氧化物酶催化与各种各样的反应,peroxidic、氧化、催化和羟基化过氧化物(如过氧化氢的存在。过氧化物酶反应可以分为四类反应[2]的基础上,氧化脱氢(2 SH +过氧化氢→2 S•+ 2水),氧化卤化(SH +过氧化氢+ H (+) + X (-)→SX + 2水,X = Cl, Br, I),过氧化氢歧化作用(2过氧化氢→2水+ O2)和传氧反应(SH +过氧化氢→SOH +水)。过氧化物酶是广泛分布在自然界中,可以很容易地提取从大多数植物细胞[3],一些动物器官和组织[4 - 7]。在工厂,他们参加木质化过程和物理防御机制受损或受感染的组织[8]。过氧化物酶是总是存在的氧化还原酶,使用过氧化氢或烷基过氧化物作为氧化剂(4、5)。许多生物物质在生化反应产生过氧化氢催化了各种bio-enzymes,所以他们最终可以确定过氧化氢的测定。近年来,提出了过氧化氢测定的各种方法,包括使用酶化验,已广泛用于分析生物化学,因为他们的速度和高选择性。辣根过氧化物酶(合)催化反应是使用最广泛的酶在生物分析化学反应[9 - 11]。酶的特点,系统地研究了过氧化氢作为氧化剂和各种物质fluorogenic基质(12-21)。 Peroxidase catalyses the various organic and inorganic substrates oxidised by one or two electron in the presence of hydrogen peroxide [21]. HRP isozyme [7] as a single chain glyco-hemoprotein, is the most abundant member of the peroxidase family [4]. It folds to an alpha-helical structure having eight helices, while the heme group (Fe (III)-protoporphyrin IX) is sandwiched between two helices. Aromatic substrates bind easily near the heme group at a specified site including Arg 38, Tyr 185, and 8-CH3 group of the pyrrole (IV) [22]. For peroxide reduction two electrons are required; one comes from Fe (III) while the other (in HRP) comes from the porphyrin, producing a porphyrin π radical cation [3, 8]. One electron reduction of compound (I) gives compound (II), in which the Fe (IV) = 0 species remains together and porphyrin is reduced [4, 23–26]. Peroxidase activity is inherent to many hemoproteins [27], such as cytochrome [28]. Peroxidases have potentially interesting application in diverse fields [5, 29–31]. In waste water treatment the aromatic phenols and amines from aqueous solutions can removed using the peroxidase/peroxide system becomes more and more interesting for biotechnologists [32] The determination of hydrogen peroxide has always been of interest because of its great importance in clinical biochemistry and also in environmental work. Several studies on the determination of hydrogen peroxide using amperometry, polarography, spectrophotometry, chemiluminescence, etc., have been published [33-37]. The instruments used in these are either very expensive or less versatile. The selectivity of the luminescence is poor. One of the drawbacks of electrochemical sensors is the interference by oxidation or reduction of other compounds at the working potential and also, electroanalytical technique needs several steps to immobilize the enzyme on a solid support, which may reduce the enzyme activity resulting in the waste of expensive biocatalyst, and it is also a time consuming process [38]. Spectrophotometric methods are simple and low cost but due to its lack of sensitivity and selectivity it is not widely used in research, environmental and clinical laboratories. We have developed a catalytic spectrofluorimetric method for the determination of POD with para-acetylaminophenol. Among the numerous methods reported in the literature for the detection of peroxidase and hydrogen peroxide, attention has chiefly been concentrated on the spectrofluorimetric method due to its highly sensitive and selective nature. The higher molar absorptivity, lower values of detection limits and RSD for HRP claims for the superiority of the method. The kinetic studies show that the lesser value of 2 2 H O m K for the peroxidase enzyme from the Lineweaver–Burk plot signifies selectivity and specificity of the proposed reaction. Para-acetylaminophenol is relatively inexpensive and water soluble. It has required sensitivity and stability. The proposed reagent, are non-carcinogenic and can replace other methods without any extra procedural difficulties as they also exhibit a good fluorescent probe.

二世。材料和方法

a装置荧光测量进行rf - 5301 pc spectrofluorophotometer日本岛津公司加上氙气光源,石英比色皿1厘米的路径长度和DR-3数据记录器。pH值的解决方案与小灵通- 4 c模型测量使用数字酸碱计在成都,中国。用于记录反应时间停表。b .试剂都是分析纯试剂的使用的所有化学品或更高等级。De-ionized水使用。0.2克毫升−1原液合被溶解适量的准备合(从Himedia实验室购买,孟买,印度)在蒸馏去离子水。过氧化氢溶液被稀释30%的解决方案准备蒸馏去离子水(标准化与高锰酸钾滴定法)。16.6毫米的磷酸二氢钾/氢氧化钠缓冲区使用pH值7.0。Para-acetylaminophenol解决方案(6.6毫米)是由溶解适量的蒸馏去离子水的试剂。c .样本和粗提物

作为过氧化物酶的来源,叶/茎部分获得马唐,胡萝卜胡萝卜,菠菜和甘蓝Oleracea Oleracea a . sessilis t . cardifolia b Oleracea var.性,var. neapolitanum,和l .漂白亚麻纤维卷收集当地市场,运输在实验室4ºC,直到使用存储在-20ºC。样品(5克)用蒸馏水洗净,均质在搅拌机使用50毫升100毫米磷酸盐缓冲剂的pH值6.0。提取是通过粗棉布和离心机12000克15分钟,上层清液是贴上粗提取液。d .蛋白测定总蛋白浓度测定一式三份的洛瑞[19]方法,使用牛血清白蛋白作为标准。大肠的动力学参数评价预测方法,单独的实验为每个过氧化氢浓度与不同浓度的Para-acetylaminophenol执行。米歇利斯- Menten常量Para-acetylaminophenol浓度从0.0618毫米到0.1545毫米。过氧化氢浓度的2.5,4.5,7.5和10毫米的最后一卷3毫升被用于每个动力学研究。pH值和温度保持不变。动力学机制之后,过氧化物酶可以证实的双重相反阴谋率与Para-acetylaminophenol浓度在不同过氧化氢浓度。假设的初始速率(¯害怕害怕一个½¯½0),机制的一般方程给出了前进的方向作为底物浓度的函数。 By rearrangement of Henri-Michaelis-Menten equation into a linear form,

F通用过程分析的过氧化物酶和过氧化氢,地方0.1毫升para-acetylaminophenol解决方案和16.6 mM KH2PO4 /氢氧化钠缓冲溶液pH值(7)为3毫升校准管,紧随其后的是0.1毫升66μM过氧化氢,用水稀释给总共3.0毫升。带温度到25±0.1¢¦C在恒温器10分钟。然后过氧化物酶的解决方案是添加为起始剂,混合大力动摇并转移到3毫升的恒温控制的细胞荧光光谱仪,它记录荧光强度的变化与时间t (F, 1 - 5分钟)λex = 326 nm和λem = 435海里。空白实验重复同样的过程获得的相对荧光强度F0ΔF的价值= F0−F计算。校准曲线是由ΔF的情节与过氧化物酶浓度的标准。同样我们发现过氧化氢的浓度。

三世。结果和讨论

a .优化实验变量的优化实验条件参数,如基质的影响,共基质、不同的缓冲的浓度,温度和潜伏期,影响酶测定,研究了。

b .光谱特性的介质的缓冲溶液pH值的荧光强度的一个重要因素被过氧化氢氧化paraacetylaminophenol的产物。我们比较KH2PO4 /氢氧化钠KH2PO4 / K2HPO4 Na3BO3-NaOH,三羟甲基氨基甲烷-盐酸缓冲液氨氯化铵缓冲和醋酸钠/醋酸缓冲与pH值控制在4.0 - -10.5范围在每种情况下,获得了不同缓冲溶液的荧光光谱。实验结果(图2 (a))显示,最佳pH值范围是7.0 KH2PO4 /氢氧化钠缓冲区。因此,pH值7.0是固定的。另外缓冲区的体积(从0.1到1.0毫升)也会影响荧光强度,不同体积的KH2PO4 /氢氧化钠缓冲溶液检查,0.1毫升的缓冲溶液3毫升(16.6毫米)反应混合物显示最大荧光强度,因此16.6毫米毫升用于后续实验。酶活性的反应对pH值如图(1)所示。

c . Para-acetylaminophenol Para-acetylaminophenol的影响效果,反应的速率进行了研究。之间存在着线性关系分析信号和para-acetylaminophenol浓度范围6.8μm 220μm,除了没有相当大的增加率。因此对于所有进一步化验para-acetylaminophenol 220μM选定的浓度。d .温度和时间对反应的影响酶的稳定性和活动主要受温度的影响。这显然可以欣赏学习时热失活的酶:酶活性随温度增加但酶稳定性降低。这些相反的趋势使温度任何酶的过程的一个关键变量,使其容易优化。温度敏感性取决于pre-incubating 3毫升的反应混合物220μM paraacetylaminophenol, 66μM过氧化氢,在16.6毫米和150点过氧化物酶KH2PO4 /氢氧化钠缓冲区在pH值为7.0 5分钟在不同温度下(0 - 80°C)。酶的活性是注册为溶液的荧光强度的函数。活动开始之后增加25°C和减少。反应时间的影响表明,氧化反应在室温下5 - 6分钟内完成; the fluorescence intensity reached a maximum after 5 min after the reagents had been added and remained constant for at least 1h. Hence, after all the oxidation reactions were carried out for 5 min, the subsequent fluorescence measurements were made at room temperature with in 1 h. E. Effect of the Addition order of Reagents Addition of various reagents in different order had influence on the fluorescence intensity. The experimental results indicated that it was optimum when solutions were added in the following order: para-acetylaminophenol, buffer, H2O2, water and peroxidase. So this order was selected in the following experiment.

f .优化过氧化氢的影响的不同浓度的过氧化氢反应速率进行了研究,在那里率线性增加66μM过氧化氢的浓度超过这个速度是独立于浓度由于酶饱和。虽然在过氧化氢的浓度越高,反应速率增加,但变化速率并不在线性范围内。因此它是决定最后的过氧化氢浓度66μM 3毫升的反应混合物。过氧化氢的反应速率的影响的插图所示图3 (B)。g .推荐过氧化物酶的测定程序0.1毫升KH2PO4 /氢氧化钠缓冲溶液pH值(7)和0.1毫升para-acetylaminophenol方案转化为10毫升校准管,紧随其后的是0.1毫升的2毫米过氧化氢,用水稀释给总共3.0毫升。把温度25±0.1一个¢¦C在恒温器5分钟。然后过氧化物酶的解决方案是添加为起始剂,混合大力动摇并转移到3毫升的恒温控制的细胞荧光光谱仪,它记录荧光强度的变化与时间t (F, 1 - 5分钟)λex = 326 nm和λem = 435海里。空白实验重复同样的过程获得的相对荧光强度F0ΔF的价值= F0−F计算。校准曲线是由ΔF的情节与过氧化物酶浓度的标准。初速度由ΔF-time记录曲线。ΔF -时间曲线的催化体系在不同浓度的合提出了如图2所示。的初始速度之间的线性关系和酶的浓度是19 - 378点。该方法的重复性检查两个系列的不同样本合19和378点的浓度,分别。 The standard deviation was 1.88 in both cases. The precession (RSD) of the fluorescence measurements was about 0.26 % in all instances.

h .量化的过氧化氢在使用之前,过氧化氢股票的解决方案是标准化与二级标准滴定KMnO4,和准确的稀释是用蒸馏水做一系列工作标准的解决方案。过氧化氢的浓度决定在3毫升的解决方案包含220μM para-acetylaminophenol解决方案,和150点过氧化物酶在16.6毫米KH2PO4 /氢氧化钠缓冲区pH值7.0。的反应是发起25°C通过添加100μL 0.5浓度线性范围内的过氧化氢。一个空白是含有水用于过氧化氢的地方。解决方案被发现spectrofluorimetrically大约5分钟后孵化(反应端点)。在选定的激发波长326 nm,我们获得了发射光谱最大相对荧光强度在435海里。初始速率当时策划对过氧化氢的浓度来获得校准图表。0.52和66.6之间的线性图的谎言μM过氧化氢。量化标定图的过氧化氢图(3)所示

即评估动力学常数建立乒乓机制和米歇利斯-基质Menten恒定值从方程(1)。最初的速度(V0)被确定为一个函数的所有基质浓度(H0 =过氧化氢,P0 = Para-acetylaminophenol)。在一个实验中,何鸿燊保持不变,当阿宝是改变,而在另一个实验中,阿宝是H0改变时保持不变。更多的实验同时Paraacetylaminophenol不同浓度的过氧化氢。常数斜率得到V0的双重相反的情节与P0(图4)在不同浓度的过氧化氢证实合的乒乓机制。拦截的re-plots图4与互惠的过氧化氢的浓度也给一个常数斜率(插图所示图。4)。KP是评估使用方程(3)发现24μM。KH的价值和Vmax线编织的过氧化物酶酶-伯克情节μM 103和1000欧盟最低为1,图5所示。

图(4):Para-acetylaminophenol为纯合的动力学行为(378点)。双倒数的情节substrate-velocity根据情商的关系。(1)。

图(5):Lineweaver-Burk情节辣根过氧化物酶的方法。动力学研究进行了378点辣根过氧化物酶。j .分析特征在最佳实验条件下,荧光强度之间的线性关系,过氧化氢浓度,在0.52 - 66μM。0.998相关系数和回归方程x是y = 6.448 + 10.28的速度法和固定时间法的相关因素是发现0.993和回归方程x是y = 10.73 + 18.74。相对标准偏差为0.26%获得一系列10标准包含150 Pico吊舱。荧光测量的标准偏差为1.88获得一系列的10个空白的解决方案。检测的限制一个¯¨LOD一个¯3½¯³坡一个¯©和检测极限一个¯¨定量限一个¯10½¯一³坡一个¯©分别是0.6995点和2.33点。k .反应机理的讨论相关的可能的反应机制是基于自我Para-acetylaminophenol耦合,一个非荧光化合物的强氧化剂如过氧化氢氧化合为一个高度荧光的荧光团2,21 -二羟基- 5,51 - diacetyldiamine联苯[39]。荧光光谱分析证实了产品的21 -二羟基- 5,51 - diacetyldiamine联苯,显示本机荧光激发最大λexc = 325 nm和排放最大λem = 439海里。

l。分析方法成功地应用于开发的应用程序在一些蔬菜样品中过氧化物酶的测定。植物提取物进行了缓冲组织比从十二1毫升g1 3:1。最高的具体活动不过是观察到9:1毫升g1比率。因此,在这个比例提取进行了系统的更好的性能。表1显示了由该方法和标准愈创木酚过氧化物酶活动方法,分光光度法。该方法的相对half-saturation点参照愈创木酚法小于1,表明更大的灵敏度的方法。量化这种方法获得的结果表明,该方法更敏感比愈创木酚的方法。植物提取物的分析从萝卜,芸苔属植物oleracea,和B。oleracea l . var.性和Daucu胡萝卜显示高过氧化物酶活动由该方法和愈创木酚法而菠菜oleracea显示少过氧化物酶活动怎么所示。

IV.CONCLUSION

没有工作到目前为止已发表的自耦合Para-acetylaminophenol量化的过氧化物酶和过氧化氢。这些共基质是多才多艺的,经济的,水溶性,有很高的催化能力和催化效率。优化的反应条件允许测定过氧化氢酶氧化低至0.52μM,哪个更敏感,也难以达到标准的愈创木酚的方法。过氧化物酶测定的线性范围的一些报道为化学发光分析方法是0.0227 - -1.136 nM[41], 5.4×身手0.1088 nM尤其对于电化学[42]方法,Paraacetylaminophenol HRP-catalyzed氧化自耦合的过氧化氢的存在允许合试验的测定的线性范围内实现18.75 - 378点。的下限检测(LOD = 0.6995 pM)和量化(2.33点)清楚地表明高灵敏度的方法。因此,该方法作为一个适当的替换为愈创木酚过氧化物酶的测定。动力学研究表明,的Km值2 2 K H O m和P m K分别为103和24.09μM。这是小比愈创木酚的方法。催化能力被发现(K P战俘)9.708×106最低为1 m - 1。由于低米歇利斯- Menten常量值,更多的催化能力提出了过氧化物酶的测定方法是更有效的天然植物提取物。

确认

报告的作者之一(Chamaraja美商)由于迈索尔大学迈索尔,卡纳塔克邦,印度提供研究设备。

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