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Functional Proteins & Genes Prac Manual7Downloaded by Seema Rayamajhi ([email protected])lOMoARcPSD|9637346
SubstrateSubstrate + enzyme(1.25 mL)(1.5 mL)Figure 4.2: When the enzyme is added to the cuvette, the substrate concentration decreasesbecause of the additional volume.For example if you have 1.25 mL of 1 mM substrate, thenafter the enzyme is added, the [S] decreases to 1.25/1.50 x 1 mM = 0.83 mM.Remember: The enzyme will not convert substrate to product until it is in added to thecuvette.Place the cuvette containing the substrate into the sample holder of the spectro.Pipette the enzyme into the cuvette, pipetting up and down a couple of times to mix theenzyme with the substrate.Shut the lid of the spectrophotometer and record absorbance.Itmay be slightly negative (why?) and record this value as 0 s and record every 15 s.3.Plot the data from all five assays on the one graph and determine Vo.Once identifying the dependant and independent variable of the experiment, plot this data ongraph paper.The units of initial velocity (Vo) are Absorbance/time.This can be obtainedfrom each graph where the enzyme is converting substrate into product at the maximum rate(i.e. the steepest part of each graph). You will need to measure the tangent of the graph atthis point to get a quantitative value for this rate. You should be able to determine 5 differentVovalues; one for each substrate concentration.4.Using the standard curve, convert Vofrom having the units of Absorbance/time to[pNP]/time.Using Vowith units of Abs/time as an unknown, determine [pNP]/time from the standardcurve.Repeat for all five Vovalues.Hint:If these values are too low to read off thestandard curve, you can use absorbance values obtained by multiplying by a constant factor,which also in used to multiple the time value.Eg If 0.05 A/15 s is too small to read off thestandard curve, this will be equivalent to 0.5 A/150 s.Reading 0.5 on they-axis maybeeasier.5.Plot [pNP]/s v [S]This is known as a Vovs [S] plot.From this graph we can get an estimate for Vmaxand Km.Estimate these values.6.Plot an Line-weaver Burk plot from the Voand [S] dataFunctional Proteins & Genes Prac Manual8Downloaded by Seema Rayamajhi ([email protected])lOMoARcPSD|9637346
Draw a table with four columns.One column will be used to indicate the [S] used andanother will be used for the [pNP]/s measured (Vo).A third column will indicate 1/[S] whilethe fourth will be 1/Vo.These two later columns will be used to plot the LB plot.You need toplot 1/Voon they-axis and 1/[S] on thex-axis.Draw a line of best fit and calculate KmandVmax.Functional Proteins & Genes Prac Manual9Downloaded by Seema Rayamajhi ([email protected])lOMoARcPSD|9637346
Figure 2.Is showing a Lineweaver Burk graph and the equation line. There’s points plottedon the line being hypothesized to aid finding Km and Vmax. The data shown in the newinverted form and a change in units shown in the corresponding columns.

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