旋光度法测定蔗糖酶促蔗糖转化反应的米氏常数实验数据处理

蔗糖α0标准浓度曲线cα0矫正0.501322.9522.890.250611.4511.390.20059.509.440.12535.805.740.10024.954.890.00000.05-0.01α∞标准浓度曲线cα∞矫正0.5-5.46-5.40.25-2.46-2.40.2-1.81-1.750.125-1.36-1.30.1-0.96-0.90-0.010.05Θ~T图0.40.60.8100200300BABNewFunction1(User)FitofSheet1BAABBStatisticsStatisticsValueStandardErrorValueStandardErrorReducedChi-SqrAdj.R-Square578.35485424.1007924.81417308.500341287.378150.80259浓度0.05时间旋光度分钟(min)秒(s)T(s)α矫正αθ2191392.152.100.8916351851.601.550.69713342141.401.350.62643562361.251.200.57334362761.151.100.53795213211.000.950.48496143740.800.750.4142Θ~T图0.30.40.50.60.7200400BABNewFunction1(User)FitofSheet1BAABBStatisticsStatisticsValueStandardErrorValueStandardErrorReducedChi-SqrAdj.R-Square-587.30141283.40519765.6761181.248331801.605540.92183浓度0.06时间旋光度分钟(min)秒(s)T(s)α矫正αθ1591191.901.850.70182441641.551.500.59863352151.251.200.51014412811.201.050.46595393391.000.750.37746444040.650.600.33327484680.600.550.31849145540.600.550.31840.00.51.005001000BABNewFunction1(User)FitofSheet1BAABBStatisticsStatisticsValueStandardErrorValueStandardErrorReducedChi-SqrAdj.R-Square771.78924116.94457188.2675653.426015456.259530.95808浓度0.08时间旋光度分钟(min)秒(s)T(s)α矫正αθ2211414.254.191.09303382183.002.940.81624222622.552.490.71665143142.502.440.7055663662.352.290.67237104301.901.840.57277594791.651.590.51738425221.301.240.43991096091.151.090.406613278070.630.570.291514468860.250.190.207415489480.200.140.1963164710070.200.140.196317471067-0.05-0.110.141018491129-0.35-0.410.074520561256-0.40-0.460.0635Θ~T图0.00.20.40.60.850010001500BABNewFunction1(User)FitofSheet1BAABBStatisticsStatisticsValueStandardErrorValueStandardErrorReducedChi-SqrAdj.R-Square854.3052759.46159266.4734326.060651790.008580.99181浓度0.10时间旋光度分钟(min)秒(s)T(s)α矫正αθ291294.003.940.86903462263.703.640.81594312713.603.540.79816303902.902.840.67417254452.602.540.62098285082.302.240.56789435832.051.990.523511276871.701.640.461413208001.201.140.372814228620.900.840.3197174410640.200.140.1956185011300.100.040.177924321472-0.30-0.360.107027591679-0.70-0.760.0361浓度0.15时间旋光度分钟(min)秒(s)T(s)α矫正αθ2351556.456.390.93364142545.855.790.8627573075.755.690.85088194994.654.590.72089135534.304.240.67941096093.903.840.632112527723.002.940.525713258052.752.690.496214138532.602.540.478416279872.302.240.4430183611161.501.440.3484221313330.800.740.2656231413940.700.640.2538272216420.150.090.188832521972-0.50-0.560.111938592339-1.05-1.110.046940172417-1.10-1.160.041044252665-1.30-1.360.0173Θ~T图0.00.51.0010002000BABNewFunction1(User)FitofSheet1BAABBStatisticsStatisticsValueStandardErrorValueStandardErrorReducedChi-SqrAdj.R-Square1169.0381851.86019391.3152319.467672461.855330.99593浓度0.30时间旋光度分钟(min)秒(s)T(s)α矫正αθ1238311.5511.490.86525233238.958.890.7114925427.557.490.628512417617.056.990.599014368766.055.990.5398165410144.704.640.4599225513753.803.740.40670.40.60.85001000BABNewFunction1(User)FitofSheet1BAABBStatisticsStatisticsValueStandardErrorValueStandardErrorReducedChi-SqrAdj.R-Square-2000.83841882.920132821.95907637.579746875.20440.96368S0ABS0/ROR00.05578.3548524.81417603.169028.28955E-050.06-587.30141765.6761178.374690.0003363710.08771.78924188.26756960.05688.33284E-050.10854.30527266.473431120.77878.92237E-050.151169.03818391.315231560.353419.61321E-050.30-2000.838412821.95907821.120660.000365354Hanes-Woolf法得截距纵坐标截距KM/rM=167.02KM=0.01774mol/L3rM=1.063*10^(-4)mol/L3【思考题】1、1、有文献指出，蔗糖转化反应的产物果糖可能对蔗糖酶产生竞争抑制作用，而葡萄糖可能产生非竞争抑制作用。试分别讨论这两种情况下，本实验采用的数据处理方法是否仍然有效？如有偏差，应如何修正？答：果糖——竞争性抑制剂通常与正常的底物或配体竞争同一个蛋白质的结合部位。这种抑制使得Km增大，而Vmax不变。对本实验数据处理有很大影响葡萄糖——抑制剂在酶的活性部位以外的部位与酶结合，不对底物与酶的活性产生竞争。抑制剂不仅与游离酶结合，也可以与酶-底物复合物结合的一种酶促反应抑制作用。酶-底物-抑制剂复合物（ESI）不能进一步释放出产物。这种抑制使得Vmax变小，但Km不变。但是需抑制剂结合的部位阻碍底物和酶的结合，即产生空间位阻也可以造成竞争性抑制。对本实验数据处理有较小的影响。2、除本实验所使用的作图法外，酶催化动力学反应数据处理还有Lineweaver－Burk作图法（双倒数作图法）、Eadie-Hofstee作图法和rm对KM作图法等，试分别用这三种方法重新计算本实验结果，并分析造成拟合相关性变差的原因。由上图可知用其他方法作图，拟合性变得很差。因为本实验上面直接拟合的参数是A和B,而由此直接得到S0/R0=A+B,后续的乘除法处理，因不同的参数数量级差异巨大，会导致当较小的数量级参于拟合时，差值分不开，导致拟合性变差。而R0本身便是较小的数量级，后面的双倒数法中1/r0就分不开差值，同理Eadie-holfstee法中，R0直接作为横坐标，也无法进行拟合。【思考及讨论】1、本实验很依赖旋光仪的读数，如果仪器本身敏感度低将会给实验结果带来巨大误差，本组所使用的旋光仪在读数过程中，由于两个三分视场之间的临界区间太大，并不可以得到准确读数，两个三分视场之间的旋光度差异可以达到10，故本次实验的原始T与σ是采用的其他组的数据，后续数据处理均为本人独立处理。2、本次实验数据的后续处理中舍弃了0.06mol和0.30mol的组别，因为该两组得到得数值异常，本身的T与σ数据取点太少，在参数A和B的拟合过程中拟合性差。