实验六__过氧化氢酶米氏常数(Km)的

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,实验七,过氧化氢酶米氏常数（,K,m,）的测定,一、实验目的,学习米氏常数（,Km,）的测定方法。,二、实验原理,过氧化氢酶,(CAT),催化下列反应：,2H,2,O,2,2H,2,O+O,2,H,2,O,2,浓度可用,KMnO,4,在硫酸存在下滴定测知。,2KMnO,4,5H,2,O,2,3H,2,SO,4,2MnSO,4,K,2,SO,4,5O,2,8H,2,O,求出反应前后,H,2,O,2,的浓度差即为反应速度。双倒数作图法求出过氧化氢酶的米氏常数。,米氏常数的测定：双倒数作图法（,Lineweaver,-Burk,法）,1,Km,1 1,=.+,v,Vmax,S,Vmax,三、试剂,1,0.05mol/L,草酸钠标准液 将草酸钠（,AR,）于,100,105,烘,12h,。冷却后，准确称取,0.67g,，用水溶解倒入,100ml,量瓶中，加入浓,H2SO4 5ml,，加蒸馏水至刻度，充分混匀，此液可贮存数周。,2,约,0.02ml KMnO4,储存液 称取,KMnO4 3.4g,，溶于,1000ml,蒸馏水中，加热搅拌，待全部溶解后，用表面皿盖住，在低于沸点温度上加热数小时，冷后放置过夜，玻璃丝过滤，棕色瓶内保存。,3,0.004mol/L KMnO4,应用液 取,0.05mol/L,草酸钠标准液,20ml,，于锥形瓶中，加浓,H2SO4 ml,，于,70,水浴中用,KMnO4,贮存液滴定至微红色，根据滴定结果算出,KMnO4,贮存液的标准浓度，稀释成,0.004mol/L,，每次稀释都必须重新标定储存液。,4,约,0.05mol/L H2O2,液 取,20%H2O2,（,AR,）,40ml,于,1000.0ml,量瓶中，加蒸馏水至刻度，临用时用,0.004mol/L KMnO4,标定之，稀释至所需浓度。,5,0.2mol/L,磷酸盐缓冲液（,pH7.0,）。,四、操作步骤,l,血液稀释 吸取新鲜（或肝素抗凝）血液,0.1ml,，用磷酸盐缓冲液,(pH7.0,，,0.2mol/L),稀释至,150ml,，得,1,：,1500,稀释血液。,2,H,2,O,2,浓度的标定：取洁净锥形瓶两只，各加浓度约为,0.05mol/L,的,H,2,O,2,2.0ml,和,25%H,2,SO,4,2.0ml,，分别用,0.004mol/L KMnO,4,滴定至微红色。从滴定用去,KMnO,4,ml,数，求出,H,2,O,2,的,mol,浓度。,3,反应速度的测定：取干燥洁净,50ml,锥形瓶,5,只，编号，按下表操作。,加入物,(ml),1,2,3,4,5,H,2,O,2,(,约,0.05mol/L),0.5,1.5,2.5,3.5,4.5,蒸馏水,4.0,3.0,2.0,1.0,血液稀释液,0.5,0.5,0.5,0.5,0.5,37,度保温,5,分钟,25,硫酸,2.0,2.0,2.0,2.0,2.0,0.004,高锰酸钾,依次加入,1,：,1500,稀释血液每瓶,0.5ml,，边加边摇，反应时间,5min,，,按顺序,向各瓶加,25%H,2,SO,4,2.0ml,，边加边摇，使酶促反应立即终止。,最后用,0.004mol/L KMnO,4,滴定各瓶至微红色，记录,KMnO,4,消耗量,(ml),。,五、计算,1.,反应速度的计算：,以反应消耗的,H,2,O,2,mmol,数表示。,反应速度,=,加入的,H,2,O,2,mmol,数剩余的,H,2,O,2,mmol,数,即：,H,2,O,2,mol,浓度,加入的毫升数,KMnO,4,0.004 mol/L,消耗的,KMnO,4,毫升数,5/2,（式中：,5/2,为,KMnO,4,与,H,2,O,2,反应中,mol,换算系数）,2,求,K,m,值：,下面引用一次实验结果为例，求过氧化氢酶的,K,m,值，供计算参考。,己知,KMnO,4,为,0.004mo1/L,，标定出,H,2,O,2,浓度为,0.05mol/L,，列表计算如下：,计算程序,1,2,3,4,5,加人,H,2,O,2,数,0.50,1.00,1.50,2.00,2.50,加人,H,2,O,2,mmol=,0.08,0.04,0.08,0.12,0.16,0.20,底物浓度,S,=,5,0.01,0.02,0.03,0.04,0.05,酶作用后，,KMnO,4,滴定,ml,1.35,3.70,6.40,9.80,13.20,剩余,H,2,O,2,mmol=,0.0045/2,0.0135,0.037,0.064,0.098,0.132,反应速度,V=,一,0.0265,0.043,0.056,0.062,0.068,按前述方法作图求得,K,m,2.5,10,-2,mol/L,。,六、思考题,l,K,m,值的意义是什么？,2,测酶,K,m,值的实验中，需要特别注意哪些操作？,