CN108717050A - 一种快速测定植物pod的方法 - Google Patents
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Abstract
本发明公开了一种快速测定植物POD的方法,包括水浴浸泡、样品研磨、酶液提取、酶活力测定,本发明的有益效果是:将传统方法,研磨效果好,特别是对于质地硬的植物组织,如种子等可达到简单、快速检测植物中的POD酶活力的目的,有利于实现批量操作。
Description
技术领域
本发明属于植物酶活力检测方法技术领域,特别涉及一种快速测定植物POD的方法。
背景技术
植物在生理代谢过程中伴随着部分电子逃逸,产生具有毒害作用的高能量活性氧,其中包括O2-、H2O2、单线态氧、羟自由基等。植物细胞的叶绿体、线粒体和质膜等是活性氧产生的主要场所。这些活性氧的积累会使植物细胞受到氧胁迫。为了减轻活性氧对细胞的破坏,维持体内活性氧的动态平衡,植物在进化过程中逐渐形成一套抗氧化酶促系统,包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)。这三种酶的作用分别是SOD催化O2-歧化反应生成H2O2,CAT分解H2O2,POD清除O2-、H2O2和其他过氧化物,与植物的生理代谢和抗逆性有密切关系。植物在生长发育过程中会受到各种不良环境的影响,包括生物胁迫和非生物胁迫。处于逆境下的植物往往会产生大量活性氧,从而也引起抗氧化酶促系统中的酶产生量发生变化以清除过多的活性氧、减少植物受到的氧胁迫。SOD、CAT和POD酶活力的变化能反映出植物所受到的胁迫状况及植物抗逆能力的强弱。因此,上述三种酶是植物生理研究中的重要研究对象,其酶活力的检测是其重要环节。
酶活力(enzyme activity)也称为酶活性,是指酶催化一定化学反应的能力。酶活力的大小可用在一定条件下,酶催化某一化学反应的速度来表示,酶催化反应速度愈大,酶活力愈高,反之活力愈低。测定酶活力实际就是测定酶促反应的速度。酶促反应速度可用单位时间内、单位体积中底物的减少量或产物的增加量来表示。
POD酶活力的检测方法:
先用冰浴研磨法和0.05mol/L pH 6.0磷酸缓冲液制备粗酶液,然后用愈创木酚法测POD酶活力;
取1g植物叶片等组织于预冷的研钵中,加入预冷的0.05mol/LpH 6.0磷酸缓冲液,研磨成浆,定容后低温离心,上清液为POD粗酶液;
愈创木酚法测定POD酶活力:酶促反应体系包含0.05mol/L pH 6.0磷酸缓冲液、0.01mol/L H2O2、0.005mol/L愈创木酚溶液和0.01-1mL酶液;将上述反应体系于波长470nm下测定吸光度的变化量,以每分钟吸光度变化0.01为一个酶活单位计算POD酶活性。
上述传统方法的缺点主要是:冰浴研磨法步骤繁琐,费时费力,批量操作困难;样品需要量较多,工作量较大。
发明内容
本发明的主要目的是针对上述现有技术中检测POD酶活力时,采用冰浴研磨法步骤繁琐、费时费力、批量操作困难,提供一种快速测定植物POD的方法,可简化操作,省时省力,并减少样品和试剂用量等。
为了实现上述发明目的,本发明采用的技术方案如下:
一种快速测定植物POD的方法,其特征是包括下述主要步骤:
(1)、水浴浸泡:
取新鲜或超低温保存的植物叶片或其它组织,在水浴中浸泡10min~20min;
(2)、样品研磨:
采用液氮研磨法进行研磨;液氮研磨法,即将样品浸入液氮后用研磨棒研磨;
(3)、酶液提取:
将上述步骤(2)研磨后的样品加入0~4℃预冷的浓度为0.1~0.4mol/L、pH6.5~7.5的磷酸缓冲液进行浸提30min~12h,0~4℃下离心处理,取上清液,POD酶液,0~4℃保存备用;
(4)、酶活力测定:
采用愈创木酚法测定POD酶活力。
所述离心,是指转速4000~16000g,时间2~15min。
与现有技术相比,本发明的有益效果是:将传统方法,研磨效果好,特别是对于质地硬的植物组织,如种子等可达到简单、快速检测植物中的POD酶活力的目的,有利于实现批量操作。
附图说明
图1示出了实施例采用本方法和传统研磨法对POD酶提取效率的影响。
具体实施方式
下面结合具体实施方式对本发明的上述发明内容作进一步的详细描述。但不应将此理解为本发明上述主题的范围仅限于下述实施例。在不脱离本发明上述技术思想情况下,根据本领域普通技术知识和惯用手段,做出各种替换和变更,均应包括在本发明的范围内。一种快速测定植物POD的方法,其特征是包括下述主要步骤:
(1)、水浴浸泡:
取新鲜或超低温保存的植物叶片或其它组织,在水浴中浸泡10min~20min;
(2)、样品研磨:
采用液氮研磨法进行研磨;液氮研磨法,即将样品浸入液氮后用研磨棒研磨;
(3)、酶液提取:
将上述步骤(2)研磨后的样品加入0~4℃预冷的浓度为0.1~0.4mol/L、pH6.5~7.5的磷酸缓冲液进行浸提30min~12h,0~4℃下离心处理,取上清液,POD酶液,0~4℃保存备用;
(4)、酶活力测定:
采用愈创木酚法测定POD酶活力。
所述离心,是指转速4000~16000g,时间2~15min。
下述各实施例中,POD酶活力的测定方法采用本的测定方法,即:
实验材料、试剂与仪器设备
1、实验仪器722型分光光度计、离心机、秒表、电子天平、研钵
2、实验试剂愈创木酚、30%过氧化氢、20mmol/LKH2PO4、100mmol/L磷酸缓冲液(pH6.0)、反应混合液[100mmol/L磷酸缓冲液(pH6.0)50mL,加入愈创木酚28uL,加热搅拌,直至愈创木酚溶解,待溶液冷却后,加入30%过氧化氢19uL,混合均匀保存于冰箱中]
3、实验材料任何植物材料
实验步骤
1、粗酶液的提取,称取植物(小麦种子),材料0.1g,加20mmol/LKH2PO45mL,在水浴中浸泡15min,再放入液氮研钵中研磨成匀浆,以10000r/min离心10分钟,收集上清液保存在冷处,所得残渣再用20mmol/LKH2PO45mL溶液提取一次,全并两次上清液。
2、酶活性的测定取比色皿2只,于一只中加入反应混合液3mL,KH2PO41mL,作为校零对照,另一只中加入反应混合液3mL,上述酶液1mL(如酶活性过高可适当稀释),立即开启秒表,于分光光度计470nm波长下测量OD值,每隔30s读数一次。以每分钟表示酶活性大小,即以△OD470/min.mg蛋白质表示,蛋白质含量测定按Folin法进行。
结果计算
以每分钟吸光度变化值表示酶活性大小,即以ΔA470/[min.g(鲜重)]表示之。也可以用每min内A470变化0.01为1个过氧化物酶活性单位(u)表示。
POD活性[u/(g.min)]=(ΔA470×VT)/(W×Vs×0.01×t)
式中:ΔA470——反应时间内吸光度的变化。W——植物鲜重,g。
VT——提取酶液总体积,mL。Vs——测定时取用酶液体积,mL。
t——反应时间,min。
Claims (2)
1.一种快速测定植物POD的方法,其特征是包括下述主要步骤:
(1)、水浴浸泡:
取新鲜或超低温保存的植物叶片或其它组织,在水浴中浸泡10min~20min;
(2)、样品研磨:
采用液氮研磨法进行研磨;液氮研磨法,即将样品浸入液氮后用研磨棒研磨;
(3)、酶液提取:
将上述步骤(2)研磨后的样品加入0~4℃预冷的浓度为0.1~0.4mol/L、pH6.5~7.5的磷酸缓冲液进行浸提30min~12h,0~4℃下离心处理,取上清液,POD酶液,0~4℃保存备用;
(4)、酶活力测定:
采用愈创木酚法测定POD酶活力。
2.如权利要求1所述的一种快速测定植物POD的方法,其特征是所述离心,是指转速4000~16000g,时间2~15min。
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