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Enzyme NANSLO Lab Activity

Page history last edited by Sue Schmidt 9 years, 7 months ago

 SUBJECT SEMESTER: XXXXXXXXXXXXXXXXXXXXX

TYPE OF LAB:  XXXXXXXXXXXXXXXXXXXX

 

 

 

 

 

TITLE OF LAB:  Enzyme

 

Enzyme NANSLO Lab Activity in Word format last updated August 22, 2014.

 

Lab format: This lab is a remote lab activity.

 

Relationship to theoryIn this lab you will be examining the underlying processes of enzyme action.

 

Instructions for Instructors:  This protocol is written under an open source CC BY license. You may use the procedure as is or modify as necessary for your class.  Be sure to let your students know if they should complete optional exercises in this lab procedure as lab technicians will not know if you want your students to complete optional exercises.

 

Instructions for Students:  Read the complete laboratory procedure before coming to lab.  Under the experimental sections, complete all pre-lab materials before logging on to the remote lab, complete data collection sections during your on-line period, and answer questions in analysis sections after your on-line period.  Your instructor will let you know if you are required to complete any optional exercises in this lab.

 

CONTENT FOR THIS NANSLO LAB ACTIVITY:

 

Learning Objectives

Background Information

Equipment

Preparing to Use the Remote Web-based Science Lab (RWSL)

Introduction to the Remote Equipment and Control Panel

Experimental Procedure

Pre-lab Exercise 1:  The Effect of Substrate Concentration on Enzyme Activity

Pre-lab Exercise 2:  The Effect of Temperature on Enzyme Activity

Exercise 1:  The Effect of Substrate Concentration on Enzyme Activity

Exercise 2:  The Effect of Temperature on Enzyme Activity

Summary Questions

 

LEARNING OBJECTIVES:

 

After completing this laboratory experiment, you should be able to do the following things:

 

  1. Define enzymes, know their functions and their characteristics.
  2. Understand how enzyme activity can be affected by certain variables.
  3. Observe and explain enzyme activity by means of a colorimetric enzyme reaction.
  4. Use quantitative data to create a graph.
  5. Determine the effect of temperature on enzymatic activity.
  6. Determine the effect of substrate concentration on enzymatic activity.
 

BACKGROUND INFORMATION:

 

Living organisms contain enzymes , which are biological catalysts used to speed up chemical reactions. Enzymes are vital to any living organisms; without their actions, chemical reactions would not occur rapidly to be able to support life.


To get a better idea of why this is important think about the process of eating a meal. We know that in a healthy diet we gain important nutrients. Nutrients are the molecules necessary for organisms to grow, reproduce and repair themselves. The breakdown of these nutrients provides energy and building blocks for living organisms via chemical reactions that will in turn result in growth, reproduction and repair.  

 

With the exception of one enzyme (the ribonuclease), all enzymes are protein molecules with specific functions and characteristics.

 

First, the basic function of any enzyme is to increase the rate of a reaction. Most cellular reactions occur about a million times faster than they would in the absence of an enzyme. Second, most enzymes act specifically with only one reactant (called a substrate) to produce products.1  So how does an enzyme work? An enzyme works by lowering the amount of energy it takes to produce a reaction, this energy is called Activation Energy – the energy required to break existing bonds to start a chemical reaction. For example to get a rock rolling down a hill, it needs a push.  That push is the activation energy.  A Catalyst is something that acts to lower the activation energy of a reaction. They reduce the “nudge” needed to get the rock rolling down the hill. Additionally, an enzyme is not used up in the reaction; it can be reused when the reaction is complete. Lastly, like all other proteins in a living cell, enzymes are produced from genes in the organism’s DNA.

 

In summary, enzymes are protein biological catalysts used by cells to drive chemical reactions.  They are reusable and are produced under the direct control of an organism’s genetic material. They work by binding to a specific molecule and putting stress on the bonds of that molecule so the reaction is more likely to occur.  If it were not for enzymes it may take weeks for our bodies to break food down.

 

How Enzymes Speed Chemical Reaction Rates: A protein based enzyme is made up of stands of amino acid chains called polypeptides.  In the process of folding these poly peptides together, folds and groves are created.  In this video https://www.youtube.com/watch?v=yZ2aY5lxEGE 2 you can get an idea of how a protein is folded to create a 3 dimensional shape.  While the video is focused on proteins it is helpful to recall that the enzymes we are working with are proteins. In an enzyme the groves created are called active sites then can react with other molecules by physically fitting together with a molecule. The molecule to which an enzyme acts upon is the substrate or reactant.  When the enzyme attaches itself to the substrate molecule, a new temporary molecule is formed the enzyme-substrate complex or ESC.  While in the enzyme substrate complex the molecules are in close proximity and the bonds are more easily altered; as a result the activation energy is less than it would be without the enzyme.   Figure 1 below shows the breakdown of a large molecule into two smaller molecules with the use of an enzyme.  Enzyme activity can be measured in the amount of product formed also known as the turnover rate.

 

Figure 1:  Formation of an Enzyme Substrate Complex and Breakdown of the SubstrateFigure 1:  Formation of an Enzyme Substrate Complex and Breakdown of the Substrate

 

Enzyme Helpers: Some enzymes use additional chemical components called coenzymes to aid in lowering the activation energy; they are non-protein organic molecules. Many of the coenzymes cannot be made by the organism, therefore must be obtained from food or are made from vitamins.  If the molecule is not organic it is called a cofactor.  These cofactors temporarily attach to the enzyme and work with the protein catalyst to speed up a reaction.

 

How the Environment Affects Enzyme Action:

  1. Temperature has two effects on enzymes:
    1. It can change the rate of molecular motion; and
    2. It can cause changes in the shape of an enzyme.  

 

                                                                            Figure 2:  Enzymes and Temperature
                                                                         Figure 2:  Enzymes and Temperature

 

Optimum temperature is the temperature at which the rate of the formation of the enzyme-substrate complex is fastest or the temperature at which the product is formed most rapidly.  When a temperature is raised above the optimum, the enzyme changes its shape so that it can no longer form the enzyme-substrate complex.   A denatured enzyme is an enzyme whose protein structure has been permanently changed so that is has lost its original biochemical properties and unable to bond properly with the substrate.

 

  1. pH The number of hydrogen ions dictate whether a pH will be acidic or basic.  The amount of hydrogen ions in an environment is very important in determining the shapes of protein molecules; too many or too few H ions can alter the shape of the enzyme.  Each enzyme has its own optimum pH where the substrates will fit; most enzymes work best in a pH at or near 7 (neutral).

                                                                                   Figure 3:  Enzymes and pH
                                                                                        Figure 3:  Enzymes and pH

  2. Enzyme-Substrate Concentration the concentration of the substrates, enzymes and products influences the rates of enzymatic reactions. If the enzyme concentration increases, the amount of product formed increases in a specific time; if the enzyme numbers are decreased, the amount of product formed decreases. If the substrate is present in a low concentration, enzyme activity will also be impacted.  Products cannot be formed until there is enough substrate and enough enzymes.

                                                                Figure 4:  Enzymes and the Effect of Concentration
                                                                        Figure 4:  Enzymes and the Effect of Concentration

 

Cellular-Controlling Processes and Enzymes: Control processes are mechanisms that ensure that an organism will carry out metabolic activities in the proper sequence (coordination) and at the proper rate (regulation).  Coordination is when the reactions take place in a specific order.  Regulation is how a cell controls the amount of chemical product produced. Below are a few of the ways this can happen.

 

  1. Gene Regulation – the number and kind of enzymes produced are regulated by the cell’s genes.  Gene regulator proteins – are chemical messengers that inform the genes as to whether they should turn enzyme production on or off. There are two types a) gene-repressor proteins decrease protein production, b) gene-activator proteins increase protein production.
  2. Feedback mechanisms – is another method to control the synthesis of molecules. Negative Feedback - As the numbers of products increase product molecules feedback to a previous reaction point in the reaction chain and have a negative effect on the enzyme controlling that reaction, thus decreasing the concentration. That is they inhibit or prevent that enzyme from performing at its best.  A good way to think of this is to compare it to how a furnace works. If the temperature drops below the set temperature the furnace kicks on and brings it back to the set temperature. An example of this in a living system is regulation of blood glucose levels.

    Positive Feedback- works in the opposite manner of negative feedback systems. As the product of an enzyme reaction increases it acts to amplify the system. So in the example of the furnace as the temperature increases to the set point on the thermostat the thermostat would respond by keeping the furnace going thus increasing the temperature even more. A biological example of this is the process of labor for child birth.
  3. Inhibition – occurs when the operation of enzymes is influenced by the presence of other molecules. An inhibitor is a molecule that interferes with an enzymes ability to form an enzyme-substrate complex; in doing so it removes a specific enzyme as a functioning part of the cell resulting in no product formation.  

 

A competitive inhibitor is any compound which closely resembles the substrate. This could be having a similar chemical composition or having a similar shape. As a result the inhibitor competes for the same active site as the substrate molecule. Competitive inhibition can be reversible if sufficient substrate molecules are available to ultimately displace the inhibitor.


A noncompetitive inhibitor is a substance that binds with the enzyme, but not necessarily at the active site.  The noncompetitive inhibitor can bind to the enzyme and cause a shape change in the active site.  As a result the substrate can no longer bind with the enzyme to cause a reaction.  A non-competitive inhibitor is usually reversible, but is not influenced by concentrations of the substrate.

Enzyme inhibition can be used to control diseases caused by bacteria by having antibiotics’ active ingredients act as the bacteria’s substrate, thus preventing the bacteria from producing the enzymes they need to function.  Some bacteria can survive and become resistant to certain antibiotics. This can occur in 4 ways:  The target is what the enzyme works on.

 

  1. They can stop producing the molecule that is the target of the drug.
  2. They can become impermeable to the drug.
  3. They can modify the target.
  4. They can release enzymes that inactivate the antibiotic.


This laboratory activity will focus on how different temperatures and different substrate concentrations affect the rate of an enzyme reaction. We will be using the enzyme glucose oxidase.

 

Glucose oxidase formula

 

Peroxidase formula

The reaction of the enzyme glucose oxidase with the β-D Glucose produces D-Gluconic acid and hydrogen peroxide (H2O2). The hydrogen peroxide then reacts with a color producing chemical to produce a color. The amount of product formed can be related to the intensity of color produced; which is then directly related to the amount of glucose used initially. The greater the color intensity, the higher the concentration of the product.  You will use a spectrophotometer to measure the absorbance. The color change we are looking for will be a pinkish color.

 

There are several relationships that enable us to measure enzyme activity using spectrophotometry. Since the rate of product formation during an enzyme-catalyzed reaction correlates with enzyme activity, and since the rate that absorbance changes during the reaction is proportional to the rate of product formation, then the rate that absorbance changes is proportional to enzymes activity.

 

Figure 5:  Spectrophotometer

 

How a Spectrophotometer works: In Figure 5 you can see the basic components of a spectrophotometer. In this simple diagram you can see the basics of how the spectrophotometer works. The light source will provide all wavelengths of visible light and wavelengths in the ultraviolet and infrared range as well. The light enters the spectrophotometer through a fiber optic port and some filters (1, 2 and 3 in the figure), and hits a series of mirrors and diffraction gratings (4, 5 and 6), which act to separate the light into its wavelengths.  The separated wavelengths are then focused through some lenses (7) onto the detector array (8) simultaneously.  The detector then sends its data to some other electronics (9 and 10), which turn the signal into the spectrum graph that you see on your computer.

 

In experiments using a spectrophotometer a blank is used in experiments. We use blanks for several reasons.  A spectrophotometer is used to measure the formation of a product as a result of a reaction. However before measuring the absorption of the product you need to know what the absorbance of the reactants are. For example in the experimental reaction we will be working with the enzyme glucose oxidase, the substrate β-D Glucose which produces D-Gluconic acid and hydrogen peroxide (H2O2). The hydrogen peroxide then reacts with a color producing chemical to produce a color. So in order determine what absorbance the product produces we need to know what the other components of the reaction absorb at.  Remember what you are looking for is the changes in the absorbance due to the substrate being changed into product. Therefore, if we can exclude these other chemicals from our spectrophotometer reading we should get clearer results. The blank is a way of doing exactly that. The blanks in this experiment include all of the reagents except the enzyme.

 

Sources:


1.    http://www.elmhurst.edu/~chm/vchembook/570enzymes.html
2.    https://www.youtube.com/watch?v=yZ2aY5lxEGE

EQUIPMENT:

 

  • Paper
  • Pencil/pen
  • Computer with Internet access (for the remote laboratory and for data analysis)

 

PREPARING TO USE THE REMOTE WEB-BASED SCIENCE LAB (RWSL):

 

For those performing a NANSLO lab for the FIRST TIME, click on this link for information on how to install software on your computer to access the RWSL interface: http://www.wiche.edu/nanslo/lab-tutorials.

 

INTRODUCTION TO THE REMOTE EQUIPMENT AND CONTROL PANEL:

 

Watch this short video to see how to use the RWSL control panel.  (SOON TO BE DEVELOPED)

There are appendices at the end of this document that you can refer to during your lab if you need to remind yourself how to accomplish some of the tasks using the RWSL control panel.

EXPERIMENTAL PROCEDURE:

 

Once you have logged on to the spectrophotometer you will perform the following laboratory procedures.

 

PRE-LAB EXERCISE 1: The Effect of Substrate Concentration on Enzyme Activity 

 

In this experiment you will be exploring looking at the effect of varying substrate concentration on the rate of the enzyme reaction. As we saw in exercise 1 enzyme activity is expressed in terms of the rate of the reaction as it is catalyzed by the enzyme. In this exercise you will collect data and then create a graph. Your graph can be used to calculate the reaction rate.


Pre-lab Questions:

 

  1. Do you think you will see a pattern in the rate of turnover as concentration changes?  Explain your answer.
  2. Do you need a new blank for this experiment? Why or why not?
  3. Hypothesis/Prediction – Set this up as an - if…..then…… statement.  For example: If heat is applied to particles in random motion then observable differences will be seen in the absorption at the different temperatures.  This example is meant to be very general; your job is to use your answer to question # 1 and make it into a more specific if-then statement based on your understanding prior to conduction the experiment.

 

PRE-LAB EXERCISE 2: The Effect of Temperature on Enzyme Activity 

 

From the background we know that temperature can have two effects on enzymes, there can be a change in the rate of molecular activity or there can be a change in the shape of the molecule.  Both of these will have an impact on turnover rate. Enzyme activity is usually expressed in terms of the rate of the reaction catalyzed by the enzyme. The rate is defined as the amount of substrate transformed, or the amount of product formed, per unit of time. We will measure the change in the absorption spectrum as either more or less product is produced.  When the data is graphed you can determine the slope of the line which will give you the reaction rate. However before we can begin you will need to determine the wavelength at which the product absorbs light maximally.


Pre-lab Questions:

 

  1. Do you think you will see a pattern in the rate of turnover as temperature changes?  Explain your answer.
  2. What color do you predict the product of the reaction will be? Defend your answer.
  3. What is the enzyme used in this experiment?
  4. What is the substrate used in this experiment?
  5. What solutions should be in the blank?
  6. Hypothesis/Prediction – Set this up as an - if…..then…… statement.  For example: If heat is applied to particles in random motion then observable differences will be seen in the absorption at the different temperatures.  This example is meant to be very general; your job is to use your answer to question # 1 and make it into a more specific if-then statement based on your understanding prior to conduction the experiment

 

EXERCISE 1: The Effect of Substrate Concentration on Enzyme Activity 

 

As with all chemical reactions whether they are mediated by biological enzymes or not the concentration of your reactants effects the reaction.  In this exercise you will be examining the effect of glucose concentration on the rate of the reaction.  For this experiment, you will want to keep the concentration of the enzyme constant while varying the concentration of glucose.  Enzyme activity is measured in unites in this experiment the stock concentration of enzyme solution is 2u /ml you will want the final concentration of the enzyme to be 0.2 u/ml.  Each cuvette used in this experiment will contain 1ml of a stock glucose solution with a concentration of 0.2 mg/ml the maximum volume of a cuvette is  4 ml.  You will need to choose four glucose concentrations between 0.1 mg/ml and 0.005 mg/ml.


Data Collection:

 

  1. Create a table and determine the volumes you are going to add of running buffer and enzyme solution.  Recorded both volumes in your table.
  2. Click the cuvette holder tab.  Set the volumes of enzyme and running buffer to match your first concentration.
  3. Set the temperature to 37°C.
  4. Select cuvette 0.
  5. Click the spectrometer tab, set the total length of time to collect data to a value between 3-5 minutes.  Set the collections interval to a value between 10-20 seconds.
  6. Set your dark and reference points.
  7. Inject your enzyme and running buffer, start recording
  8. Click the spectrometer tab and export your data.
  9. Repeat steps 1-8 with cuvette 1 and your second concentration.
  10. Repeat steps 1-8 with cuvette 2 and your third concentration.
  11. Repeat steps 1-8 with cuvette 3 and your fourth concentration.


Analysis:

 

  1. Create a data table that shows the concentration, absorbance and time.
  2. Using the data create a graph. On the graph you will plot time as the independent variable and absorbance as the dependent variable. You should have three different lines for the concentration ratio variables.
  3. With your graphed data calculate the slope of the line for the last 5 minutes of the data collected at each concentration. Slope is calculated by first choosing two points on the graph then determining the change in the horizontal points and the change in the vertical points. For example if your coordinates on the graph at point 1 are 1(x axis) and 6 (y axis) and at point 2 they coordinates are 4(x axis) and 12 (y axis). So the vertical change is 12 - 6 and the horizontal change is 4 – 1 or vertical change = 6 and horizontal change = 3. Then to find the slope you would divide the vertical change by the horizontal change. 6/3 for a slope of 2. Be sure to include the correct units for your data. What information can you get from this graph?
  4. In what way is the rate of the reaction dependent on the concentration of the substrate? How would this change if we were testing the concentration of the enzyme?
  5. In the experiments with a high substrate concentration, the absorbance will likely flatten out near the end of the experiment. If the glucose oxidase is not a reversible enzyme, what do you believe is the reason for the flattening out of the curve?
  6. Based on the data you collected write a claim/evidence statement. In other words make a claim based on what you learned in this experiment and back it up with the data you collected.

 

EXERCISE 2: The Effect of Temperature on Enzyme Activity 

 

You are measuring reaction rates (change in product concentration per unit time). Therefore, it is important that you take your absorbance measurements at the precise time intervals. 

          
In addition, you must keep in mind that even when you are taking your absorbance measurement the reaction will still be proceeding, and more enzymatic product will be forming.


Do not wait for the numbers to stop changing—they won’t!


This also means that you should not “pause” your timing of the reaction when you are taking your readings from the spectrophotometer.  

 
Data Collection:

 

  1. Click the cuvette holder tab.  Set the volumes of enzyme and running buffer to match your fastest reaction in exercise 1.
  2. Set the temperature to 10°C.
  3. Select cuvette 4.
  4. Click the spectrometer tab, set the total length of time to collect data to a value between 3-5 minutes.  Set the collections interval to a value between 10-20 seconds.
  5. Set your dark and reference points.
  6. Inject your enzyme and running buffer start recording
  7. Click the spectrometer tab and export your data.
  8. Repeat steps 1-7 at 70°C.


Analysis:

 

  1. Create a data table that shows the temperature, absorbance and time at 10°C, 37°C (from exercise 1), and 70°C.
  2. Using the data create a graph. On the graph you will plot time as the independent variable and absorbance as the dependent variable. You should have three different lines for the temperature variables.
  3. With your graphed data calculate the slope of the line for the last 5 minutes of the data collected at each temperature. Slope is calculated by first choosing two points on the graph then determining the change in the horizontal points and the change in the vertical points. For example if your coordinates on the graph at point 1 are 1(x axis) and 6 (y axis) and at point 2 they coordinates are 4(x axis) and 12 (y axis). So the vertical change is 12 - 6 and the horizontal change is 4 – 1 or vertical change = 6 and horizontal change = 3. Then to find the slope you would divide the vertical change by the horizontal change. 6/3 for a slope of 2. What information can you get from this graph?
  4. Analyze the graph by relating the shape of the curve to an underlying mechanism that might govern the phenomenon being studied. Discuss factors involved in enzyme action that might cause this curve shape to be true.  
  5. On your time versus absorbance graph interpolate what the absorbance would be at 35°C if you took a reading at 5 minutes?
  6. On your time versus absorbance graph extrapolate after 30 minutes what would the absorbance be at your highest temperature? Would the graph continue to rise? Explain your reasoning.
  7. How is the activity of the enzyme affected by temperature?
  8. Based on the data you collected write a claim/evidence statement. In other words make a claim based on what you learned in this experiment and back it up with the data you collected.

 

SUMMARY QUESTIONS: 

 

  1. Why do you need to establish the wavelength for maximal absorption first?
  2. In these experiments is the blank the same thing as a control?
  3. Explain what the absorbance will tell you about the activity of the enzyme.
  4. Predict what would happen if the enzyme was boiled for 30 minutes then reacted with the substrate. Do some research to explain and defend your answer.
  5. If given enough time, will all of the experimental tests reach the same absorbance?  Why?
  6. If you vary pH, how do you think this will affect an enzyme? Why?
  7. Explain the role of enzymes inside our body. Provide examples of enzymes.


Choose two of the following research questions to answer:

  1. Research enzyme inhibition and write out a paragraph describing a method of testing for enzyme inhibition.
  2. Research and choose an enzyme related genetic disorder. Write a short summary of the enzyme affected and how it impacts the body.
  3. Research and explain how the three-dimensional shape of an enzyme is determined?
  4. Lactose-intolerance is a common condition among several people worldwide. Using reliable scientific resources, research and write in a short paragraph about this condition.
  5. Most viruses (with the exception of a few) lack basic metabolic enzymes but yet they manage to infect our body and lead to serious diseases and infections. Discuss how they are capable of doing all chemical reactions inside the host cells.
  6. Imagine you are working in international research laboratory as a prominent research scientist. Knowing that each enzyme has a set of optimal conditions (pH, temperature). Describe how you will go about to find out the optimal conditions for a newly discovered enzymes.

 

 

For more information about NANSLO, visit www.wiche.edu/nanslo.

 

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Creative Commons Attribution 3.0 United States License 3  

 

 

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