最新植物生理指标测定方法

最新植物生理指标测定方法 实验一 植物叶绿素含量的测定(分光光度法) (张宪政，1992) 一、原理 根据叶绿体色素提取液对可见光谱的吸收，利用分光光度计在某一特定波长测定其吸光度，即可用公式计算出提取液中各色素的含量。根据朗伯—比尔定律，某有色溶液的吸光度A与其中溶质浓度C和液层厚度L成正比，即A,αCL式中:α比例常数。当溶液浓度以百分浓度为单位，液层厚度为1cm时，α为该物质的吸光系数。各种有色物质溶液在不同波长下的吸光系数可通过测定已知浓度的纯物质在不同波长下的吸光度而求得。如果溶液中有数种吸光物质，则此混合液在某一波长下的总吸光度等于各组分在相应波长下吸光度的总和。这就是吸光度的加和性。今欲测定叶绿体色素混合提取液中叶绿素a、b和类胡萝卜素的含量，只需测定该提取液在三个特定波长下的吸光度A，并根据叶绿素a、b及类胡萝卜素在该波长下的吸光系数即可求出其浓度。在测定叶绿素a、b时为了排除类胡萝卜素的干扰，所用单色光的波长选择叶绿素在红光区的最大吸收峰。高等植物中叶绿素有两种:叶绿素a 和b，两者均易溶于乙醇、乙醚、丙酮和氯仿。叶绿素a和叶绿素b的比值反映植物对光能利用效率的大小，比值高则大，则反之。 二、、仪器设备及试剂 试剂:1)95%乙醇(或80%丙酮) 三、实验步骤 称取剪碎的新鲜样品 0.2,0.3g，加乙醇10ml，提取直至无绿色为止。把叶绿体色素提取 液倒入光径1cm的比色杯内，以95%乙醇为空白，在波长663nm和645nm下测定吸光度。 四、实验结果按计算 丙酮法(Arnon法)【可以用于丙酮乙醇混合法和80%丙酮提取法的计算】 叶绿素a的含量(mg/g) =(12.71，OD663 – 2.59，OD645)V/1000*W 叶绿素b的含量(mg/g) =(22.88OD645 – 4.67OD663) V/1000*W 叶绿素a、b的总含量(mg/g) =(8.04，OD663 +20.29，OD645) V/1000*W 按Inskeep公式 叶绿素a的含量(mg/g) =(12.63，OD663 – 2.52，OD645)V/1000*W 叶绿素b的含量(mg/g) =(20.47OD645 – 4.73OD663) V/1000*W 叶绿素a、b的总含量(mg/g) =(7.90，OD663 + 17.95，OD645) V/1000*W 注:1、叶绿素a和叶绿素b的比值反映植物对光能利用率 【1】 比如阳生植物叶绿素a和叶绿素b的比值较大 【2】阴生植物叶绿素a和叶绿素b的比值较小 2、丙酮-------熔点:-94? ;沸点:56.48?;是一种无色透明液体，有特殊的辛辣气味易溶于水和甲醇、乙醇、乙醚、氯仿、吡啶等有机溶剂. 下一步实验方法比较 【1】95%乙醇直接提取(?) 【2】95%乙醇加热提取(冯瑞云，1985) 【3】无水酒精和80%丙酮等体积混合提取 实验二、不良环境对植物细胞膜的伤害((张宪政，1992)) 一、 原理 植物组织在受到各种不利的环境条件(如干旱、低温、高温、盐渍和大气污染)危害时，细胞膜的结构和功能首先受到伤害，细胞膜透性增大。若将受伤害的组织浸入无离子水中，其外渗液中电解质的含量比正常组织外渗液中含量增加，组织受伤害越严重，电解质含量增bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 加越多。用电导仪测定外渗液电导率的变化，可反映出质膜受伤害的程度。在电解质外渗透的同时，细胞内可溶性有机物也随之渗出，引起外渗液可溶性糖、氨基酸、核苷酸等含量增加，氨基酸和核苷酸对紫外光有吸收，对紫外分光光度计测定受伤害组织外渗液消光值，同样可反映出质膜受伤害的程度。用电导仪法和紫外法测定结果有很好的一致性。 二、方法步骤 1、取样及处理 采用电导法(张宪政，1992):将选取的幼嫩叶片和成熟叶片剪下，包在密封袋内置于冰盒中带回实验室。将新鲜的叶样先用自来水轻轻冲洗叶片，除去面沾污物，再用去离子水冲洗1~2次，用滤纸轻轻吸干叶片表面水分，称0.2,0.3 g并剪成切段，放入50 mL带塞试管中，加入20 mL 去离子HO，浸没样品4~5 h，各处理浸泡时间和测定温度一致，在室温下2 进行，用DDs?11型电导仪测其电导率，然后沸水浴15 min，冷却至室温再测一次总电导率值。以相对电导率表示细胞质膜透性大小。 2、计算: 植物组织浸泡的电导率，特称外渗电导率，此测定方法称外渗电导率法。 (1)外渗电导率 K(us/cm*g) =SQ=测定电导率/称取质量 或 K(us/cm*g*ml)=SQ=测定电导率/称取质量*量取体积 (2)相对电解质渗出率. 电解质渗出率(%)= L1(浸泡液电导率)/ L2(煮沸后电导率)×100 电解质渗出率(%)= L1(浸泡液电导率)-本底电导率/[ L2(煮沸后电导率)-本底电导率]×100 式中:L1:组织杀死前外渗液的电导值;L2组织杀死后外渗液的电导值。 注:1、事先测定水的电导率 2、用吸水纸吸干探头的水分 实验三 植物体内游离脯氨酸含量的测定 一、目的 在逆境条件下(旱、热、冷、冻)，植物体内脯氨酸的含量显著增加，植物体内脯氨酸含量在一定程度上反映了植物的抗逆性，抗旱性强的品种积累的脯氨酸多。因此测定脯氨酸含量可以作为抗旱育种的生理指标。另外，由于脯氨酸亲水性极强，能稳定原生质胶体及组织内的代谢过程，因而能降低冰点，有防止细胞脱水的作用。在低温条件下，植物组织中脯氨酸增加，可提高植物的抗寒性，因此，亦可作为抗寒育种的生理指标。 二、原理 磺基水杨酸对脯氨酸有特定反应，当用磺基水杨酸提取植物样品时，脯氨酸便游离于磺基水杨酸溶液中。然后用酸性茚三酮加热处理后，茚三酮与脯氨酸反应，生成稳定的红色化合物，再用甲苯处理，则色素全部转移至甲苯中，色素的深浅即表示脯氨酸含量的高低。在520nm波长下测定吸光度，即可从曲线上查出脯氨酸的含量。 三、材料、仪器及试剂 1.试剂及配制: ,1(1)2.5,酸性茚三酮溶液配制:将1.25g茚三酮溶于30ml冰醋酸和20ml 6mol?L , 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" topeoplehe theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 rom the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and thip, fe current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationsr to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on thn ordeond battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations iassigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the secadres headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cth. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental e Sou, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in thscary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South seems uiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities sufferedion in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Hmbers on the party organizatbridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party meople [in the tomb of the stadium (now the tongluo secondaryhool were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the pe' sc Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls 2 磷酸中(原液稀释2.3倍)，搅拌加热(70?)溶解，贮于4?冰箱中，2-3日有效。 (2)3,磺基水杨酸配配制:3.496g磺基水杨酸加蒸馏水溶解后定容至100ml。 (3)10μg?ml-1脯氨酸标准母液配制:精确称取20mg脯氨酸，倒入小烧杯内，用少量蒸馏水溶解，再倒入200ml容量瓶中，加蒸馏水定容至刻度(为100μg?ml,1脯氨酸母液)，再吸取该溶液10ml， 加蒸馏水稀释定容至100ml，即为10μg?ml-1脯氨酸标准液。 四、实验步骤 1、脯氨酸标准曲线的制作 1.1取6支试管，编号，按下表配制每管含量为0,12μg的脯氨酸标准液。加入表中试剂后，置于沸水浴中加热30min。取出冷却。以去离子水溶液为空白对照，在520mm波长处测定吸光度(A)值。 管 号 试 剂 0 1 2 3 4 5 10μg?ml-1脯氨酸标准液(ml) 0 0.2 0.4 0.6 0.8 1.0 蒸馏水(ml) 2 1.8 1.6 1.4 1.2 1.0 冰醋酸(ml) 2 2 2 2 2 2 3%磺基水杨酸 2 2 2 2 2 2 2.5,酸性茚三酮(ml) 4 4 4 4 4 4 每管脯氨酸含量(μg) 0 2 4 6 8 10 1.3标准曲线的绘制 以1,5号管脯氨酸含量为横坐标，吸光度值为纵坐标，绘制标准曲线。 样品的测定 2. 2.1脯氨酸的提取 称取0.2,0.3g叶片于20ml试管 + 10ml 3,磺基水杨酸溶液?沸水浴10min(提取过程中要经常摇动)?冷却过滤于干净的试管中?脯氨酸提取液。 2.2测定 吸取酶提取液2ml + 2ml HO + 2ml冰醋酸 + 4ml 2.5,酸性茚三酮?沸水浴30min?溶2 液呈红色?冷却?取上层液于比色杯520mm处测定吸光度(A)值。 五、计算结果 从标准曲线上查出样品测定液中脯氨酸的含量，按下公式计算样品中脯氨酸含量: X×提取液总量(ml) ,1脯氨酸含量(μg?gFw), ——————————————————— 样品鲜重(g)× 测定时提取液用量(ml) 公式中:X ,从标准曲线中查得的脯氨酸含量(μg) 实验四 植物体内丙二醛含量的测定 一、原理 丙二醛(MDA)是由于植物官衰老或在逆境条件下受伤害，其组织或器官膜脂质发生过氧化反应而产生的。它的含量与植物衰老及逆境伤害有密切关系。测定植物体内丙二醛含量，通常利用硫代巴比妥酸(TBA)在酸性条件下加热与组织中的丙二醛产生显色反应，生成红棕色的三甲川(3、5、5,三甲基恶唑2、4,二酮)，三甲川最大的吸收波长在532nm。但是测定植物组织中MDA时受多种物质的干扰，其中最主要的是可溶性糖，糖与硫代巴比妥酸显色反 so on;rets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and e enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secapaneshe masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Jigher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing tnext hrce controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the on, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire folidatis actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consonity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang'portuno. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the op . Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating andembers. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party moon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local peoplebridge, in the response from the crowd was great, and Wang sthe tongluo secondary(now ed down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadiumool was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burny sch8 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementarthere are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1933 应产物的最大吸收波长在450nm处，在532nm处也有吸收。植物遭受干旱、高温、低温等逆境胁迫时可溶性糖增加，因此测定植物组织中丙二醛与硫代巴比妥酸反应产物含量时一定要排除可溶性糖的干扰。此外在532nm波长处尚有非特异的背景吸收的影响也要加以排除。低浓度的铁离子能显著增加硫代巴比妥酸与蔗糖或丙二醛显色反应物在532、450nm处的吸光度值，所以在蔗糖、丙二醛与硫代巴比妥酸显色反应中需要有一定的铁离子，通常植物组织中铁离子的含量为100,300μg?g,1Dw，根据植物样品量和提取液的体积，加入Fe3，的终浓度为0.5nmol? L,1。在532nm、600nm和450nm波长处测定吸光度值，即可计算出丙二醛含量。 二、实验步骤 (1)提取 采用硫代巴比妥酸显色法(赵世杰等，2002)。称取0.2,0.3 g样品，加10%三氯乙酸2 mL和少量石英砂，研磨至匀浆，再加8ml 三氯乙酸进一步研磨，匀浆后4000 rpm，离心10min。 (2)显色 取上清液5 mL，加0.6% TBA 5 mL，混合后在100?水浴上煮沸30 min，迅速在冰浴上冷却后再离心一次。分别测定上清液在450nm、532 nm和600 nm处的吸光度值， 方法一: MDA质量摩尔浓度(nmol/g)=( A532-A600)×V×V/(0.155×W×V) Z12 式中:V: 反应液总量(4 mL); V1: 提取液体积(10 mL); Z V: 测定用用提取液量(2 mL); W: 取样叶鲜重(g) 2 0.155:为MDA的消光系数(nmol/l) 方法二: 方程组: A450=(85.4×10,3)?C糖 (A532,A600)=(155×10,3)?CMDA+(7.4×10,3)?C糖 解得: A450 C糖= —————— = 11.71A450(mmol?L,1) 85.4×10,3 C= 6.45(A532,A600),0.56A450(μmol?L,1) MDA 公式中:A450—在450nm波长下测得的吸光度值 A532—在532nm波长下测得的吸光度值 A600—在600nm波长下测得的吸光度值 , —1.55×105为摩尔比吸收系数 C糖、CMDA分别是反应混合液中可溶性糖、MDA的浓度。 1. 按下式计算提取液中MDA浓度 反应液体积(ml) C × ————————— MDA 1000 提取液中MDA浓度(μmol?ml,1)= ——————————————— 测定时提取液用量(ml) 2.按下式计算样品中MDA含量 e current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationsr to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on thn ordeond battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations iassigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the secadres headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cth. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental e Sou, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in thscary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South seems uiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities sufferedion in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Hmbers on the party organizatbridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party meople [in the tomb of the stadium (now the tongluo secondaryhool were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the pe' sc Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls , 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" topeoplehe theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 rom the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and thip, f4 提取液中MDA浓度(μmol?ml,1)×提取液总量(ml) MDA含量(μmol?g,1 Fw)= ———————————————————————— 植物组织鲜重(g) 试剂: 1、10,三氯乙酸:称取三氯乙酸55.5556g加水溶解定容至500ml 2、0.6,硫代巴比妥酸(用10,三氯乙酸配制):称0.6707g TBA用少量1mol/L氢氧化 钠溶解后加10,TCA溶解定容至100ml(4?贮存) 实验五、SOD活力测定(NBT还原法) 植物叶片在衰老过程中发生一系列生理生化变化，如核酸和蛋白质含量下降、叶绿素降解、光合作用降低及内源激素平衡失调等。这些指标在一定程度上反映衰老过程的变化。近来大量研究表明，植物在逆境胁迫或衰老过程中，细胞内自由基代谢平衡被破坏而有利于自由基的产生。过剩自由基的毒害之一是引发或加剧膜脂过氧化作用，造成细胞膜系统的损伤，严重时会导致植物细胞死亡。自由基是具有未配对价电子的原子或原子团。生物体内产生的自由基主要有超氧自由基(O2,)、羟自由基(OH.)、过氧自由基(ROD)、烷氧自由基(RO)等。植物细胞膜有酶促和非酶促两类过氧化物防御系统，超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(ASA-POD)等是酶促防御系统的重要保护酶。抗坏血酸(VC)、VE和还原型谷胱甘肽(GSH)等是非酶促防御系统中的重要抗氧化剂。SOD、CAT等活性氧清除剂的含量水平和O2,、H2O2、OH. 和O2等活性氧的含量水平可作为植物衰老的生理生化指标。 超氧自由基(O2.,)是生物细胞某些生理生化反应常见的中间产物。自由基是本身带有不成对价电子的分子、原子、原子团或离子，化学性质非常活泼，是活性氧的一种。如果细胞中缺乏清除自由基的酶时，机体就会受到各种损伤。超氧化物歧化酶(Superoxide Dismutase)，简称SOD，能通过歧化反应清除生物细胞中的超氧自由基(O2.,)，生成H2O2 。H2O2由过氧化氢酶(CAT)催化生成H2O和O2，从而减少自由基对有机体的毒害。 和O2 一、原理 超氧物歧化酶(superoxidedismutase，SOD)普遍存在于动、植物体内，是一种清除超氧阴离子自由基的酶。本实验依据超氧物歧化酶抑制氮蓝四唑(NBT)在光下的还原作用来确定酶活性大小。在有氧化物质存在下，核黄素可被光还原，被还原的核黄素在有氧条件下极易再氧化而产生O2-，可将氮蓝四唑还原为蓝色的甲腙，后者在560nm处有最大吸收。而SOD可清除O2-，从而抑制了甲腙的形成。于是光还原反应后，反应液蓝色愈深，说明酶活性愈低，反之酶活性愈高。据此可以计算出酶活性大小。 二、试剂 (一)试剂 (1)0.2 mol/L pH7.8磷酸钠(Na2HPO4-NaH2PO4)缓冲液: A液(0.2 mol/L NaH2PO4溶液):准确称取NaH2PO4?2H2O (MW=156.01)15.715g于50mL小烧杯中，用少量蒸馏水溶解后 ，移入500mL容量瓶中用蒸馏水定容至刻度，充分混匀。4?冰箱中保存备用。 B液(0.2 mol/L Na2HPO4溶液):准确称取Na2HPO4?12H2O(MW=358.14)71.63g于100mL小烧杯中，用少量蒸馏水溶解后，移入1000mL容量瓶中用蒸馏水定容至刻度，充分混匀。4?冰箱中保存备用。 取上述A液34mL与B液366mL充分混匀后即为0.2mol/L pH7.8的磷酸钠缓冲液。4?冰箱中保存备用。 (2)0.05 mol/L pH7.8磷酸钠缓冲液 igher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing tnext hrce controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the on, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire folidatis actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consonity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang'portuno. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the op . Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating andembers. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party moon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local peoplebridge, in the response from the crowd was great, and Wang sthe tongluo secondary(now ed down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadiumool was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burny sch8 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementarthere are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 193 so on;rets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and e enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secapaneshe masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate J5 取0.2 mol/L pH7.8的磷酸钠缓冲液250mL，移入1000 mL容量瓶中用蒸馏水定容至刻度，充分混匀。4?冰箱中保存备用。 (3)130mmol/L 蛋氨酸(Met)磷酸钠缓冲液 准确称取L-蛋氨酸(C5H11NO2S，MW=149.21)0.9699g于小烧杯中，用少量0.05 mol/L pH7.8的磷酸钠缓冲液溶解后，移入50mL容量瓶中并用0.05 mol/L pH7.8的磷酸钠缓冲液定容至刻度，充分混匀(现用现配)。【溶于水，3.3g/100ml 25?，不溶于有机溶剂】4?保存可用1,2d. (4)750μmol/L 氮蓝四唑溶液:称取0.03066g NBT用磷酸缓冲液定容至50ml，避光4?保存，先用现配. (5)100μmol/L EDTA,Na2溶液:称取0.03721g EDTA,Na2用水定容至1000ml【称取7.442 g EDTA,Na2用水定容至200ml，吸取1ml用水定容1000ml】 (6)20μmol/L 核黄素溶液:称取0.0753g核黄素用蒸馏水定容至1000ml避光保存【称取1.8825g核黄素用少量NaOH溶解并用蒸馏水定容至25ml，吸取1ml用水定容1000ml】。4?保存8—10天。 三、实验步骤 1. 酶液提取 取植物叶片0.2g,0.3g于预冷的研钵中，加2ml预冷的0.05 mol/L pH7.8磷酸缓冲液在冰浴上研磨成浆，加缓冲液使终体积为10ml。于4000rpm下离心15min，上清液即为SOD粗提液。 2. 显色反应 取试管3支，1支为测定管，另2支为对照管，按下列加入一依次加入各溶液: 名称 样品管 对照管1 对照管2 50mmol/L 磷酸缓冲液(ml) 5.0 5.0 5.0 130mmol/L Met溶液(ml) 0.3 0.3 0.3 750μmol/L NBT溶液(ml) 0.3 0.3 0.3 100μmol/L EDTA,Na2液(ml) 1.0 1.0 1.0 去离子水(ml) 2.0 2.0 2.0 20μmol/L 核黄素(ml) 0.3 0.3 0.3 0.3(酶液) 0.3(缓冲液) 0.3(缓冲液) 混匀后将1支对照管置暗处，其它各管于4000Lx日光下反应20min，然后立即遮光，以遮光管为对照调零，于560nm下测定光密度。(要求各管受光情况一致，温度高时间缩短，低时延长)。 3.SOD活性测定与计算 至反应结束后，以不照光的对照管做空白，分别测定其它各管的吸光度。 四、结果计算 已知SOD活性单位以抑制NBT光化还原的50,为一个酶活性单位表示，按下式计算SOD活性。 SOD总活性,(Ack,AE)×V/(Ack×0.5×W×Vt) =(Ack,AE)/(Ack×0.015×W) SOD比活力,SOD总活性/蛋白质含量 上式中:SOD总活性以每克鲜重酶单位表示; SOD比活力:单位以酶单位,mg蛋白表示 Ack:照光对照管的吸光度 AE:样品管的吸光度 V:样品液总体积(ml) Vt测定时样品用量(ml) he theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 rom the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and thip, fe current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationsr to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on thn ordeond battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations iassigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the secadres headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cth. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental e Sou, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in thscary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South seems uiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities sufferedion in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Hmbers on the party organizatbridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party meople [in the tomb of the stadium (now the tongluo secondaryhool were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the pe' sc Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls , 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" topeople6 实验六、过氧化物酶活性测定 一、试验目的: 过氧化物酶是植物体内普遍存在的活性较高的一种酶，他与呼吸作用、光合作用以及生长素的氧化等都有密切关系。在植物生长发育过程中他的活性不断发生变化，因此测量这种酶，可以反映某一时期植物体内代谢的变化。通过本实验了解过氧化物酶的作用，掌握常用的测定过氧化物酶的方法——愈创木酚法。 二、实验原理: 在过氧化物酶催化下，双氧水将愈创木酚氧化成茶褐色产物，此产物在λ470 nm处有最大吸收峰，故可以通过测其吸光度的变化测定过氧化物酶的活性。 三、实验试剂 1、0.05 mol/L PH 7.0的磷酸缓冲液: 0.2M A液(Na2HPO4)61ml和0.2M B液(NaH2PO4)39ml混合100ml，用去离子水定容400ml 2、0.05 mol/L愈创木酚溶液:取0.6207g定容100ml 1%愈创木酚溶液:吸取1毫升愈创木酚，加入少量95%酒精溶解，用水定容100ml，放于棕色试剂瓶中保存备用 3、2%双氧水溶液::取30% HO 10ml用去离子水定容150ml 22 4.、反应混合液 取50mmol/L磷酸缓冲液(pH7.0)30mL，2%过氧化氢10mL，1%愈创木酚10mL混合。 四、操作步骤 1(酶液提取:清洗干净叶片擦干，称取0.2,0.3 g叶片，剪成小片，放入研钵加入适量10%PVPP(除去酚类物质)、石英沙、及2ml 50mmol的PH 7.0(5.5-7.5)的磷酸缓冲液PBS(0.02-0.1mmol)进行研磨，磨碎后在加入8mlPBS溶液洗涤研钵一并装入离心管内，4?下 -14000rmin离心15min，收集上清液4?保存备用。 2(显色反应 类别 对照管 样品管 反应液 3.0 mL 25? 温度 缓冲液0.5 mL 酶液0.5 mL 测定波长 470nm 五、计算 过氧化物酶活性(?OD470/(min.g)) =(?A470×VT)/(W×VS×0.01×t) 式中:?A470为反应时间内吸光度的变化，W为叶片鲜重g，t为反应时间min，Vt为提取酶液总体积ml，Vs为测定时取用的酶液体积ml。 the tongluo secondary(now ed down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadiumool was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burny sch8 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementarthere are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 193 so on;rets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and e enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secapaneshe masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Jigher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing tnext hrce controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the on, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire folidatis actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consonity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang'portuno. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the op . Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating andembers. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party moon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local peoplebridge, in the response from the crowd was great, and Wang s7 实验19植物组织中可溶性蛋白质含量的测定 ? 考马斯亮蓝 G – 250 染色法 一、原理 考马斯亮蓝 G – 250 ( Coomassie brilliant blue ， G-250 )法是利用蛋白质 – 染料结合的原理，定量地测定微量蛋白质浓度的快速、灵敏的方法。 考马斯亮蓝 G-250 存在着两种不同的颜色形式，红色和蓝色。它和蛋白质通过范德瓦尔键结合，在一定蛋白质浓度范围内，蛋白质和染料结合符合比尔定律。此染料与蛋白质结合后颜色由红色形式转变成蓝色形式，最大光吸收由 465 nm 变成 595 nm ，通过测定 595 nm 处光吸收的增加量可知与其结合蛋白质的量。 蛋白质和染料结合是一个很快的过程，约 2 min 即可反应完全，呈现最大光吸收，并可稳定 1 h ，之后，蛋白质–染料复合物发生聚合并沉淀出来。此法灵敏度高(比 Lowry 法灵敏 4 倍)，易于操作，干扰物质少，是一种比较好的定量法。其缺点是在蛋白质含量很高时线性偏低，且不同来源蛋白质与色素结合状况有一定差异。 二、实验材料、试剂与仪器设备 (一)试剂 1. 标准蛋白质溶液(100 μg/mL 牛血清白蛋白):称取牛血清蛋白25 mg ，加水溶解定容至 100 mL ，吸取上述溶液40 mL ，用蒸馏水稀释至100 mL 即可。 并 2. 考马斯亮蓝试剂:称取 100 mg 考马斯亮蓝 G-250 ，溶于 50 mL 90 ,乙醇中，加入 100 mL 85,( W/V)的磷酸，再用蒸馏水定容到 1000 mL ，贮于棕色瓶中。常温下可保存一个月。 三、实验步骤 1. 标准曲线的绘制 取 6 支试管，按表 26–1 加入试剂，摇匀，向各管中加入 5 mL 考马斯亮蓝试剂，摇匀，并放置 5 min 左右，以 0 号试管为空白对照，在 595 nm 下比色测定吸光度。以蛋白质含量为横坐标，以吸光度为纵坐标绘制标准曲线。 管 号 试 剂 0 1 2 3 4 5 标准蛋白质( mL ) 0 0.20 0.40 0.60 0.80 1.00 蒸馏水量( mL ) 1.00 0.80 0.60 0.40 0.20 0 蛋白质含量( μg ) 0 20 40 60 80 100 2. 样品测定 ( 1 ) 样品提取 称取鲜样 0.25 , 0.5 g ，用 5 mL 蒸馏水或缓冲液研磨成匀浆后， 3000 r/min 离心 10 min ，上清液备用。 ( 2 )吸取样品提取液 0.3 mL (视蛋白质含量适当稀释)，放入试管中(每个样品重复 2 次)，加入 5 mL 考马斯亮蓝试剂，摇匀，放置 2 min 后在 595 nm 下比色，测定吸光度，并通过标准曲线查得蛋白质含量。 ond battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations iassigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the secadres headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cth. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental e Sou, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in thscary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South seems uiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities sufferedion in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Hmbers on the party organizatbridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party meople [in the tomb of the stadium (now the tongluo secondaryhool were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the pe' sc Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls , 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" topeoplehe theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 rom the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and thip, fe current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationsr to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on thn orde8 四、结果计算 ( mg/g ) 式中: C ——查标准曲线值， μg 。 V T ——提取液总体积 , mL 。 W F ——样品鲜重 , g 。 V S ——测定时加样量 , mL 。 注意点: 1，考马斯亮蓝G-250所配溶液不稳定，所以每次实验都必须新配，且必须新做标准曲线; 2，考马斯亮蓝G-250配制完毕，如果发现溶液中有絮状沉淀，可以试用滤纸过滤后使用，如果实验中发现使用过滤后的考马斯亮蓝G-250溶液在与样品接触后短时间内便又发生絮凝，基本上推测该考马斯亮蓝G-250过期(比较容易出现)。 实验七、过氧化氢酶的活性测定——高锰酸钾滴定法 【原理】 过氧化氢酶(CAT)属于血红蛋白酶，含有铁，它能催化过氧化氢分解为水和分子氧，在此过程中起传递电子的作用，过氧化氢则既是氧化剂又是还原剂。 据此，可根据HO的消耗量或O的生成量测定该酶活力大小。在反应系统中加入一定222 量(反应过量)的HO溶液，经酶促反应后，用标准高锰酸钾溶液(在酸性条件下)滴定多22 余的HO22 5HO+2KMnO+4HSO5O+2KHSO+8HO+2MnSO 22424 2424 即可求出消耗的HO的量。 22 【试剂】 【1】3M HSO:吸取浓硫酸80ml用去离子水定容至500ml 24 【2】0.2mol/L磷酸缓冲液pH7.0; 【3】0.1mol/L高锰酸钾标准液:称取KMnO(AR)16 g，用新煮沸冷却蒸馏水配制成4 1000ml，用0.1mol/L草酸溶液标定; 0.1mol/L HO:市售30% HO大约等于17.6mol/L，取30% HO溶液4.87ml，稀释至222222500ml，用标准0.1mol/ KMnO溶液(在酸性条件下)进行标定; 4 【实验步骤】 1、酶液提取 取叶片0.2,0.3g加入2ml pH7.0的磷酸缓冲液少量研磨成匀浆，在加缓冲液定容转移至10ml后转入离心管中，4000rpm离心15min，上清液即为过氧化氢酶的粗提液。 (now ed down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadiumool was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burny sch8 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementarthere are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 193 so on;rets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and e enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secapaneshe masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Jigher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing tnext hrce controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the on, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire folidatis actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consonity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang'portuno. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the op . Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating andembers. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party moon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local peoplebridge, in the response from the crowd was great, and Wang sthe tongluo secondary9 2.滴定反应 管 号 对照管 样品管 粗酶液(ml) 0.2 0.2 3mol/L HSO (ml) 5 0 24 PH 7.0磷酸缓冲液 1.8 1.8 0.1mol/L HO (ml) 5 5 22 条 件 加入HO立即计时于35?恒温水浴中保温3min 22 3mol/L HSO (ml) 0 5 24 用0.1mol/L KMnO标准溶液滴定HO，至出现粉红色(在30min内不消失)为终点 422 所用KMnO体积(ml) A B 4 【结果计算】: 酶活性用每克鲜重样品1min内分解HO的毫克数表示: 22 ().ABV,，，T17 酶活(mgHO/gFW?min)= 22 1FWVt，， 式中 A—对照KMnO滴定毫升数; 4 B—酶反应后KMnO滴定毫升数; 4 V—酶液总量(ml); T V—反应所用酶液量(ml); 1 W—样品鲜重(g); 1.7—1ml 0.1mol/L的KMnO相当于1.7mg HO。 422 过氧化氢酶的活性测定——紫外吸收法 一、原理 HO在240nm波长下有强烈吸收，过氧化氢酶能分解过氧化氢，使反应溶液吸光度(A)24022 随反应时间而降低。根据测量吸光率的变化速度即可测出过氧化氢酶的活性。 二、实验步骤 1、酶液提取 取叶片0.2,0.3g加入2ml pH7.0的磷酸缓冲液少量研磨成匀浆，在加缓冲液定容转移至10ml后转入离心管中，4000rpm离心15min，上清液即为过氧化氢酶的粗提液。4?下保存备用。 2.测定:取10ml试管3支，其中1号为样品测定管，1号为空白管，按表40-2顺序加入试剂。 表40-2 紫外吸收法测定HO样品液配置表 22 管 号 样品管 对照管 粗酶液(ml) 0.2 (煮死酶液)0.2 pH7.0磷酸(ml) 1.5 1.5 蒸馏水(ml) 1.0 1.0 25?预热后，逐管加入0.5ml 0.1mol/L的HO，每加完一管立即记时，并迅速倒入石22 英比色杯中，240nm下测定吸光度，每隔1min读数1次，共测4min，待3支管全部测定完后，按下式计算酶活性。 rom the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and thip, fe current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationsr to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on thn ordeond battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations iassigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the secadres headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cth. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental e Sou, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in thscary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South seems uiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities sufferedion in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Hmbers on the party organizatbridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party meople [in the tomb of the stadium (now the tongluo secondaryhool were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the pe' sc Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls , 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" topeoplehe theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 10 3.结果计算: 以1min内A减少0.1的酶量为1个酶活单位(u)。 240 A，V240,T过氧化氢酶活性(u/gFW/min)= 0.1，V1，t，FW ，()AASS12式中 A= A, ,240 S02 A—加入煮死酶液的对照管吸光值; S0 ASAS—样品管吸光值; Vt—粗酶提取液总体积(ml); ， 1 2 V1—测定用粗酶液体积(ml); FW—样品鲜重(g); 0.1—A每下降0.1为1个酶活单位(u); t—加过氧化氢到最后一次读数时间(min)。 240 高锰酸钾滴定液的配制和标定 高锰酸钾滴定液的配制和标定 1(高锰酸钾滴定液(0.1mol,L)的配制 市售高锰酸钾常含有少量MnO等杂质，蒸馏水也常有微量的灰尘，溶液在配制初期不2 够稳定，浓度常降低，因此高锰酸钾溶液不能用直接法配制，所以先配制成近似所需的浓度。 【1】方法:称取稍多于理论用量的固体高锰酸钾，用新煮沸并冷却的蒸馏水溶解，放置两天以上或加热至沸，并保持微沸15min，使各种还原性物质完全氧化，用垂熔玻璃器过滤，除去MnO等沉淀，摇匀，储存于棕色瓶中，暗处密闭保存。 2 【2】操作步骤:称取高锰酸钾16g，加蒸馏水1000ml，煮沸15min、放冷，转人棕色瓶中密闭避光保存，2天后过滤待标定。 2(高锰酸钾滴定液(0.1mol,L)的标定 基准物: NaCO 224 在硫酸(0.5,lmol,L)溶液中，加热至75,85C时反应如下: +2+-- 2MnO，5CO，16H?2Mn，10CO?，8HO 42422 但溶液温度低于60?，反应速度较慢，如温度高于90?时，则草酸钠会分解，使测定结果偏高。 操作步骤:称取恒重的基准物NaCO约0.2g，加新煮沸过的冷蒸馏水20ml、3mol/L的 224 HSO10ml使溶解，加热至75?，自滴定管中逐滴加人待标定的高锰酸钾溶液，滴定至溶液24 显微红并保持30秒不褪色为终点。当滴定完成时，溶液温度应不低于55?。 CV=2/5 *(m/M) 高高草草 e enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secapaneshe masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Jigher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing tnext hrce controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the on, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire folidatis actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consonity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang'portuno. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the op . Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating andembers. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party moon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local peoplebridge, in the response from the crowd was great, and Wang sthe tongluo secondary(now ed down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadiumool was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burny sch8 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementarthere are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 193 so on;rets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and 11 过氧化氢配制和标定 过氧化氢配制和标定 一、实验原理 酸性介质、室温下: 5HO+2KmnO+4HSO?5O?+2KHSO+MnSO+8HO 224242442 KMnO为自身指示剂，终点颜色:微红色(半分钟内不裉色) 4 二、实验步骤 1(HO溶液的配制 22 移取HO溶液4.9 mL置于500mL容量瓶中，加水稀释，定容，摇匀。 22 2(HO含量的测定 22 .-1移取配制得到的HO溶液20.00mL于锥形瓶中，加入3molLHSO 5mL，用KMnO标22244准溶液滴定到溶液呈微红色，半分钟不褪色即为终点。平行三次。 计算公式: CV=5/2 *CV 过过高高 备注 备注 一、磷酸缓冲液 储备液A:0.2M Na2HPO4溶液(53.65g Na2HPO4*7HO或71.7g Na2HPO4*12H2O配成1000ml) 2 储备液B:0.2M NaH2PO4溶液(27.8g NaH2PO4*H2O配成1000ml) ,ml A +,mlB，稀释至200ml.即为0.1M磷酸缓冲液 pH值 0.2mol/LNa2HPO4溶液(mL) 0.2mol/LNaH2PO4溶液(mL) 5.7 6.5 93.5 5.8 8.0 92.0 5.9 10.0 90.0 6.0 12.3 87.7 6.1 15.0 85.0 6.2 18.5 81.5 6.3 22.5 77.5 6.4 26.5 73.5 6.5 31.5 68.5 6.6 37.5 62.5 6.7 43.5 56.5 ond battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations iassigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the secadres headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cth. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental e Sou, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in thscary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South seems uiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities sufferedion in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Hmbers on the party organizatbridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party meople [in the tomb of the stadium (now the tongluo secondaryhool were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the pe' sc Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls , 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" topeoplehe theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 rom the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and thip, fe current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationsr to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on thn orde12 6.8 49.0 51.0 6.9 55.0 45.0 7.0 61.0 39.0 7.1 67.0 33.0 7.2 72.0 28.0 7.3 77.0 23.0 7.4 81.0 19.0 7.5 84.0 16.0 7.6 87.0 13.0 7.7 89.5 10.5 7.8 91.5 8.5 7.9 93.5 7.5 8.0 94.7 5.3 ed down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadiumool was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burny sch8 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementarthere are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 193 so on;rets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and e enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secapaneshe masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Jigher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing tnext hrce controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the on, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire folidatis actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consonity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang'portuno. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the op . Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating andembers. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party moon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local peoplebridge, in the response from the crowd was great, and Wang sthe tongluo secondary(now 13