!!! Cautions for this lab:
1.O-dianisidine dye is carcinogenic and toxic. Avoid contact with your skin, wash your hands after use, and notify your instructor immediately if a spill occurs.
2.Do not add the diluted enzyme until you are ready to start the reaction. This is time = 0!
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Enzyme extract dilution
Tube # / Dilution / Vol Enzyme / Vol Water1 / ------ / 1.0ml extract / ------
2 / 1:2 / 1.0ml extract / 1.0ml
3 / 1:10 / 200μl extract / 1.8ml
4 / 1:100 / 20μl extract / 1.98ml
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Identification of good enzyme dilution (the good dilution of enzyme should give 0.4-0.7 absorbance units after 5 minutes). Use this good dilution in all of three of your enzyme experiments. For each sample (except the blank) in the following four tables, ten data have to be collected (Read the OD460nm value every 30 seconds for five minutes).
Cuvette label / Vol pH7 buffer / Vol Dyeo-dianisidine / Vol H2O2 / When ready to run reaction, add Vol extract
D1 / 2.4ml / 50μl / 0.5ml / 50μl 1:100 dilution(#4)
D2 / 2.4ml / 50μl / 0.5ml / 50μl 1:10 dilution(#3)
D3 / 2.4ml / 50μl / 0.5ml / 50μl 1:2 dilution(#2)
D4 / 2.4ml / 50μl / 0.5ml / 50μl undiluted(#1)
B (blank) / 2.4ml / 50μl / 0.5ml water / 50μl 1:10 dilution(#3)
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For the experiments below, read the absorbance (460nm) every 30 seconds for 5 minutes.
Part III: effect of pH
Cuvette label / Vol buffer / Vol Dyeo-dianisidine / Vol H2O2 / When ready to run reaction, add Vol extract
B (blank) / 2.4ml / 50μl / 0.5ml water / 50μl good dilution
pH2 / 2.4ml / 50μl / 0.5ml / 50μl good dilution
pH4 / 2.4ml / 50μl / 0.5ml / 50μl good dilution
pH7 / 2.4ml / 50μl / 0.5ml / 50μl good dilution
pH8 / 2.4ml / 50μl / 0.5ml / 50μl good dilution
pH10 / 2.4ml / 50μl / 0.5ml / 50μl good dilution
Part I: effects of the substrate
Cuvette label / Vol pH 7 buffer / Vol dyeo-dianisidine / Vol water / Vol H2O2 / When ready to run reaction, add extract
S1 / 2.4ml / 50μl / 0.48ml / 20μl / 50μl good dilution
S2 / 2.4ml / 50μl / 0.45ml / 50μl / 50μl good dilution
S3 / 2.4ml / 50μl / 0.40ml / 100μl / 50μl good dilution
S4 / 2.4ml / 50μl / 0.30ml / 200μl / 50μl good dilution
S5 / 2.4ml / 50μl / 0.20ml / 300μl / 50μl good dilution
S6 / 2.4ml / 50μl / 0.10ml / 400μl / 50μl good dilution
S7 / 2.4ml / 50μl / ------ / 500μl / 50μl good dilution
B (blank) / 2.4ml / 50μl / 0.5ml / ------ / 50μl good dilution
Part II: effects of inhibitors
Cuvette label / Vol pH 7 buffer / Vol dyeo-dianisidine / Vol water / Vol H2O2 / Vol inhibitors / When ready to run reaction, add extract
I1 / 2.4ml / 50μl / 0.48ml / 20μl / 10μl / 50μl good dilution
I2 / 2.4ml / 50μl / 0.45ml / 50μl / 10μl / 50μl good dilution
I3 / 2.4ml / 50μl / 0.40ml / 100μl / 10μl / 50μl good dilution
I4 / 2.4ml / 50μl / 0.30ml / 200μl / 10μl / 50μl good dilution
I5 / 2.4ml / 50μl / 0.20ml / 300μl / 10μl / 50μl good dilution
I6 / 2.4ml / 50μl / 0.10ml / 400μl / 10μl / 50μl good dilution
I7 / 2.4ml / 50μl / ------ / 500μl / 10μl / 50μl good dilution
B’ (blank) / 2.4ml / 50μl / 0.5ml / ------ / 10μl / 50μl good dilution
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Calculation instructions (only for reference, units&scales on the graph may vary!!)
Part I: effects of the substrate
Seven ABSORBANCE460nm vs TIME plots (S1-S7) on the same set of axes with appropriate legend. There should be seven lines in one graph.
Find out the slope for each line (there should be seven slopes), that is your ΔOD/min, use Beer-Lambert law: OD=ε×c×l to calculate Δc/min, that is your velocity V0 (ε=11.3mM-1cm-1, l =1cm).
Now draw Michaelis-Menten and Lineweaver-Burke graphs (there should be seven dots on each of the plot). From Lineweaver-Burke graph, you can find out the Vmax and Km based on the y-intercept and x-intercept.
Remember the concentration of H2O2 solution is 8.8mM, so you have to calculate the [S] according to the dilution you did in the cuvettes.
Part II: effects of inhibitors
Similar to Part I, seven ABSORBANCE460nm vs TIME plots (I1-I7) on the same set of axes with appropriate legend.There should be seven lines in one graph.
Find out the slope for each line (there should be seven slopes), that is your ΔOD/min, use Beer-Lambert law: OD=ε×c×l to calculate Δc/min, that is your velocity V0 (ε=11.3mM-1cm-1, l =1cm).
Now draw Michaelis-Menten and Lineweaver-Burke graphs on the same corresponding graph in Part I (there should be seven dots on each of the plot). From Lineweaver-Burke graph, you can find out the Vmax and Km based on the y-intercept and x-intercept. Based on the Lineweaver-Burke (double reciprocal) plot, decide what type of inhibition do your inhibitors cause? (Competitive, uncompetitive, or non-competitive?)
Remember the concentration of H2O2 solution is 8.8mM, so you have to calculate the [S] according to the dilution you did in the cuvettes.
Part III: effect of pH
Five ABSORBANCE460nm vs TIME plots (pH2-pH10) on the same set of axes with appropriate legend. There should be five lines in one graph.
Find out the slope for each line (there should be seven slopes), that is your ΔOD/min, use Beer-Lambert law: OD=ε×c×l to calculate Δc/min, that is your velocity V0 (ε=11.3mM-1cm-1, l =1cm).
Plot V0 vs pH like the graph below. There should have five dots on the graph, after put the five dots on the graph connect them together with a smooth curve.
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What is expected in your lab report?(Each group only needs to give me one report, so collaborate with your group members.)
1. All of your absorbance data.
2. All the slope values you find from the ABSORBANCE460nm vs TIME graphs and all your calculated data for the Michaelis-Menten graph and Lineweaver-Burke graph.
3. Totally three ABSORBANCE460nm vs TIME graphs (part I&II should have seven lines on each graph; part III should have five lines on it).
4. One Michaelis-Menten graph and one Lineweaver-Burke graph (each one of the graphs should combine both part I&II results which means there should be two curves on the Michaelis-Menten graph and two lines on the Lineweaver-Burke graph) Besides Lineweaver-Burke graph, you should put the calculated Km and Vmax values (for with inhibitor only).
5. V0vs pH graph for part III.
6. For part II, how has the line changed on Michaelis-Menten graph due to the inhibitor? What type of inhibition do your inhibitors cause based on this plot?
7. For part III, at what pH is peroxidase most active?
8. Did you find anything unexpected in your experiments? If so, what is/are the mostly likely reason(s)?
All the graphs and data analysis MUST be done with one of the softwares, hand-drawn graphs are NOT acceptable! Here are some recommended softwares: Microsoft Excel, Sigmaplot, SPSS, OriginPro, GraphPad Prism, etc.. Any software in any platformis FINE, as long as the software can clearly present and analyze your data.
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