In this activity, you will study the effect of pH, temperature and lactose concentration on lactase activity.  By the end of this activity, you should be able to:

design experiments to study the effects of pH, temperature, and substrate concentration on enzyme activity.

graph data related to enzyme activity.

analyze data related to enzyme activity.

                identify the components of the scientific method.

                identify components of the substrate-enzyme-product system being studied.

                summarize and communicate your lab data.

To prepare for the lab, watch the following video and think about enzymes, substrates, products, maximum rate of reaction, and optimal conditions.  https://www.youtube.com/watch?v=D2j2KGwJXJc  

To complete the lab activity, watch the Zoom link for lab posted on Canvas and go to the following website (https://sites.google.com/site/biologydarkow/lactase-enzyme-simulation

   Read through the Home, Background and Explore the Model tabs on the website. 

Experiment I:  The effect of pH on lactase activity.   Click on the Simulate tab.  The standard condition settings are:   lactose for 500 mg/dL , pH at 7.0 and temperature at 250C and 5mmol lactase enzyme solution.  You will vary one of these conditions you wish to study in each experiment.  The condition you are varying is the independent variable for that experiment.  The outcome you measure for that experiment is the dependent variable.  The hypothesis of the experiment is a tentative proposed relationship of the independent and dependent variable of the experiment.  In this experiment, you are studying pH. 

  1.  Determining the inflection point and interval of interest.  Before you can begin your experiment, you must determine the interval of pH which will give you the most valuable, relevant data.  Start by running a simulation with the standard initial lactose, temperature and lactase enzyme solution.  Run simulations at a pH of 3.  Record the glucose concentration (mg/dL) at 4, 7 and 11 minutes.  Repeat the simulations at a pH of  5, 7, 9, and 11.  Record the glucose concentration (mg/dL) at 4, 7 and 11 minutes. 
pH4 minutes7 minutes11 minutes
3   
5   
7   
9   
11   

Produce a graph using the above data with all three time lines (4, 7, and 11 minutes) for the data you used to find the inflection point on the same graph (See the Zoom recording for this lab).   Watch the following videos for some suggestions.https://www.youtube.com/embed/3PwVWX28dEE?feature=oembedhttps://www.youtube.com/embed/Vk8F99Ptq2E?feature=oembed

  1. Use the inflection you determined above to give you the pH interval which will give you the most valuable, relevant data for determining the optimum pH.   Divide this interval into ten (10) equally spaced intervals  (for example if you determine the interval between pH 7 and 11 is where the peak enzyme rate is occurring, you would collect data at 7.4, 7.8, 8.2, 8.6 …. 10.2, 10.6 and 11).  Develop a data collection table to record the results of these simulations at 4 minutes, 7 minutes and 11 minutes for each pH level.  Run the simulation and record the data.  Produce line graphs using your data.
pH4 minutes7 minutes11 minutes
    
    
    
    
    
    
    
    
    
    

Produce three individual graphs using the data you collected within the interval of interest:   1) pH : glucose concentration 4 minutes; 2) pH : glucose concentration 7 minutes and;  3) pH : glucose concentration 11 minutes.  Look at the graphs and determine the optimum pH (where maximum lactase enzyme activity is likely located). 

Experiment II:  The effect of temperature on lactase activity.   Use the Experiment I to design an experiment to test the effect of temperature on lactase activity.  What is the independent variable of the temperature experiment?   What is the dependent variable of the temperature experiment?  Which variables will you standardize in the temperature experiment? 

I.  Describe the experiment you designed to determine the inflection point and interval of interest for temperature. 

II.  Describe the experiment you designed to determine the optimum temperature.  Use the tables to record and four graphs to report your data.  What is the optimum temperature for lactose?

  1.  Determine the inflection point and interval of interest for temperature.
Temperature (C)4 minutes7 minutes11 minutes
    
    
    
    
    

After you complete the table, use the data to produce a graph with all three times (4, 7, and 11 minutes) on the same graph.  What is the interval of interest and inflection point based on your results?  The interval of interest is the range of temperatures which will give you the most valuable, relevant data for determining the optimum temperature in the second part of the experiment.

  1. Use the inflection and temperature interval of interest to design the second part of the experiment.  Divide this interval into ten (10) equally spaced intervals.  Use the table below to record the results of these simulations at 4 minutes, 7 minutes and 11 minutes for each temperature.  Run the simulation and record the data. 
Temperature (C)4 minutes7 minutes11 minutes
    
    
    
    
    
    
    
    
    
    

Use the data from the table to produce three individual graphs:   1) temperature : glucose concentration at 4 minutes. 2) temperature : glucose concentration at 7 minutes, and 3)  temperature : glucose concentration at 11 minutes.  Look at the graphs and determine the optimum temperature (where maximum lactase enzyme activity is likely located).   

Experiment III:  The effect of lactose concentration on lactase activity.   For this experiment, use the first two experiments as models for studying the effect of lactose concentration on lactase activity.  Note that you will need to develop your own data collection tables to record data and graphs to report your results.  Design an experiment to test the effect of lactose concentration on lactase activity.  What is the independent variable of this experiment?   What is the dependent variable of this experiment?  Which variables will you standardize?  Describe your experiments.  What is the optimum lactose concentration for maximum lactase activity?

Experiment IV:  Studying specificity using lactase activity.   Are you able to design an experiment to study specificity in lactase using the Lactase Enzyme Simulation? Yes or No   Explain specificity and the experiment you would design to study this concept in lactase using the simulation or how you would need to modify the simulation to test this concept.  You don’t need to do this experiment, just explain its design.

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