ENYZYME LAB Effects of Temperature and pH on Enzyme Function Anatomy & Physiology Instructor Terry Wiseth Northland College Click To Go To Next Slide

Click To Go To Next Slide ENZYME LAB Directions in performing the lab In the performance of this virtual lab understanding how the buttons will work for you should be helpful. Navigating the lab will be performed by clicking on the various buttons available in a menu you will find in the lower right hand corner. Click on the YELLOW button below to go to the next slide and learn how to navigate. Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Click To Go To Next Slide Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will allow you to proceed to the next slide. You will use this button often. Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will allow you to go to the previous slide in the lab. Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will allow you to go to the slide that you have just came from. Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will allow you to quit the lab and go back to your desktop. Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will bring you to the lab index. From the lab index you can link to either Lab #1 or Lab #2. Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will bring you to Lab #1 Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will bring you to Lab #2 Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Click Here If You Understand How To Use All Of These Buttons ENZYME LAB Click on each of the buttons that you see below to view description of how each button works Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show Clicking this button will bring you to the Enzyme Lab Tutorial. The lab tutorial provides more information about enzymes. Click Here If You Understand How To Use All Of These Buttons Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

ENZYME LAB Click To Go To Next Slide This button is utilized to go to the next slide of the lab. This would be analogous to turning the page of your lab book Click To Go To Last Slide Viewed This button is utilized to return to the slide that you linked from initially. This would be analogous to using a book mark in your lab book This button is utilized to link to your lab report. Using this button will bring you to pages that should be printed out so that you can include them in your lab report Click To Go To Lab Report You probably want to utilize this button next

Click to View Data Table Click To Go To Conversion Chart ENZYME LAB This button is utilized to give information on when or how to use a specific piece of lab apparatus Turn a Machine On Click to View Data Table These buttons are utilized to go to examples of graphs or tables which may be more helpful if you print them off Click to View Graph Click To Go To Conversion Chart This button is utilized to link to conversion charts which are helpful in entering the correct values in the data tables You probably want to utilize this button next

ENZYME LAB You should print off the following pages located from these links Density Conversion Chart Temperature Hypothesis pH Hypothesis Temperature Data Table pH Data Table Graph of Temperature vs mM product Graph of pH vs mM product Temperature Conclusion pH Conclusion Temperature Lab Questions pH Lab Questions

ENZYME LAB This ENZYME LAB is divided into two Experiments Experiment #1 will investigate the influence temperature has on enzyme activity Experiment #2 will investigate the influence pH has on enzyme activity

ENZYME LAB INDEX Click To Go To The Enzyme Tutorial (Learn More About Enzymes) Click To Go To Experiment #1 Effect of Temperature on Enzyme Activity Go Back to A&P 2 Lab Book Click To Go To Experiment #2 Effect of pH on Enzyme Activity

Temperature Influences ENZYME LAB #1 Temperature Influences On Enzyme Activity

ENZYME LAB #1 INDEX Lab # 1 Discussion How To Use The Spectrophotometer How To Use The Temperature Probe How To Use The Hot Plate Start Of Lab # 1 Exercise Links To Charts And Graphs

ENZYME LAB The following is what you should make sure that you include in your lab report 1) Hypothesis 2) Data Table 3) Graph (mM Product vs Temperature) 4) Conclusion 5) Answers to questions 1 - 4

ENZYME LAB Changes in temperature can dramatically influence the activity of most enzymes by affecting enzyme structure As temperatures increase enzyme activity is greater due largely to the increase in kinetic energy (energy of motion) that allows the enzyme and substrate to collide with higher frequency

ENZYME LAB Once the temperature optimum is reached, enzyme activity decreases slowly as the enzyme is heated because the enzyme is undergoing denaturation Denaturation is a term used to describe a change in shape occurring to enzymes

ENZYME LAB When an enzyme’s shape is changed it can no longer perform its function as efficiently Just like a wrench that has lost its shape when over-heated in a forge, an enzyme is not able to function when it has lost its shape

ENZYME LAB This exercise is designed to help you understand the effect of temperature on enzyme activity You will also analyze data from this experiment to determine the ideal temperature optimum for Invertase (an enzyme) activity

ENZYME LAB To begin the experiment we will determine the temperature of the substrate The substrate is the chemical material which the enzyme will be acting on

ENZYME LAB As is the case with real experiments in a biochemistry lab, enzyme should always be added to the substrate last to prevent the reaction from starting before all necessary components have been added

Click To Go To Lab Report Hypothesis ENZYME LAB The first thing you will do is to develop a hypothesis to predict the effect of an increase in temperature on invertase activity Enter your hypothesis in the first line of your lab report A hypothesis is a best guess at what you think may be the results of the experiment Click To Go To Lab Report Hypothesis

ENZYME LAB The substrate used in this experiment is Sucrose Substrate concentration for sucrose for this experiment is 90 millimolar units (mM)

ENZYME LAB The enzyme used in this experiment is Invertase Invertase will react with sucrose in a biochemical reaction that results in the formation of by-products Invertase sucrose (+ water)              glucose + fructose Sucrase

ENZYME LAB Accumulation of by-products in the solution over time will create a change in the relative density of the solution This relative density can be measured by the use of a Spectrophotometer ON 4000 Photons

HOW TO USE THE SPECTROPHOTOMETER ENZYME LAB HOW TO USE THE SPECTROPHOTOMETER ON 4000 Photons

The spectrophotometer is designed to measure the amount of light which is entering into the device The device records a measurement in photons which can be extrapolated into a measure of density ON 4000 Photons Spectrophotometer

In the beakers below and to the left is a series of solutions with varying degrees of density When an enzyme such as invertase is added to a sucrose solution the resulting enzymatic reactions cause an accumulation of by-product ON 4000 Photons Spectrophotometer

This accumulation of product results in an increase in density of the solutions By placing the beakers in front of a Spectrophotometer we are able to measure the relative density of each solution ON 4000 Photons Spectrophotometer

Click on the top beaker (lightest color) which appears to have the least density The beaker will now be placed in front of the spectrophotometer ON 4000 Photons Spectrophotometer

Turn the spectrophotometer on by clicking the ON button to measure the light intensity which is passing through the beaker The measurement you see on the readout from the Spectrophotometer is in photons and will need to be converted to relative density ON 4000 3200 Photons Spectrophotometer

Click To Go To Conversion Chart Click on the GREEN button below the table to go to the chart which will convert the measurement of PHOTONS into units of DENSITY The DENSITY units can be correlated to mM of product produced and are the values which you will use to graph your results ON 3200 9000 Photons Spectrophotometer Click To Go To Conversion Chart

Click To Go To Conversion Chart Click on the second beaker which appears to have an intermediate density and then click the ON button on the spectrophotometer Use the conversion chart again to convert photons to density by clicking on the GREEN button below the table ON 2800 4000 Photons Spectrophotometer Click To Go To Conversion Chart

Click To Go To Conversion Chart Click on the third beaker which appears to have the highest density and then click the ON button on the spectrophotometer Use the conversion chart again to convert photons to density by clicking on the GREEN button below the table ON 2600 4000 Photons Spectrophotometer Click To Go To Conversion Chart

HOW TO USE THE TEMPERATURE PROBE ENZYME LAB HOW TO USE THE TEMPERATURE PROBE 30o 35o Temp

Temperature Probe 30o 35o Sucrose Solution Hot Plate A Temperature Probe can be used to measure the temperature of the sucrose solution A Temperature Probe is inserted into the solution which will give measurements of centigrade degrees Temperature Probe 30o 35o Temp H C Sucrose Solution Hot Plate

Temperature Probe 30o Sucrose Solution Hot Plate Click on the beaker containing the solution to set it on the Hot Plate Now click on the Temperature Probe to insert it into the solution Record the measure of the temperature of the solution Temperature Probe 30o Temp H C Sucrose Solution Hot Plate

ENZYME LAB HOW TO USE THE HOT PLATE H C

Temperature Probe 30o 35o Sucrose Solution Hot Plate A Hot Plate is used to heat the solution Temperature can be controlled by using the dial on the side of the Hot Plate Be very careful not to touch the Hot Plate with your hands or you may burn yourself Temperature Probe 30o 35o Temp H C Sucrose Solution Hot Plate

Temperature Probe 30o 35o Hot Plate Click the RED KNOB on the Hot Plate to increase the amount of heat Again insert the Temperature Probe into the solution by clicking on it and record the temperature Temperature Probe 30o 35o Temp H C Hot Plate

Temperature Probe Spectrophotometer 9000 3200 30o 35o Hot Plate Familiarize yourself again with each of the instruments below Insert the Temperature Probe, Turn the Spectrophotometer on, and Turn up the heat on the Hot Plate for practice ON Temperature Probe Spectrophotometer 9000 3200 Photons 30o 35o Temp H C Hot Plate Insert Temperature Probe by clicking on it Click red knob to start hot plate Turn Spectrophotometer On Return to Index

Click To Go To Experiment #1 You are now ready to begin the experiment to see how temperature effects the rate of enzyme action Click on the Yellow button below to go to experiment #1 ON 4300 9000 Photons 30o 35o Temp H C Click To Go To Experiment #1

Click To Go To Data Table In the beaker below is a 90 mM sucrose solution which will act as the substrate The temperature probe will be used to measure the temperature of the substrate solution before heating it with the Hot Plate Click on the temperature probe to measure the temperature of the substrate and then record your results in the data table under temperature for measurement #1 Measurement # 1 30o Temp Click To Go To Data Table

Click To Go To Conversion Chart Click on the beaker with the 90 mM sucrose solution to place it in front of the Spectrophotometer Next turn the Spectrophotometer on and record your data on your data sheet ON 3600 Photons Measurement # 1 Spectrophotometer Click To Go To Conversion Chart

ENZYME LAB At this point you should have recorded the first line of data on your data sheet for experiment #1 Data Table Measurement Temperature Photons mM of Product 1 30 3600 2 3 4 5 6 7 8 9 10 11

Sucrose solution Invertase Next we will add an enzyme to the substrate solution (Sucrose) The enzyme we will use is called INVERTASE Invertase is an enzyme which will metabolize or break down sucrose When sucrose is broken down the build up of metabolic by-products causes changes in the density of the sucrose-invertase solution Sucrose solution Invertase

In the beaker below is a 90 mM sucrose solution In the test tube to the right is an enzyme called invertase Pour the invertase into the sucrose solution by clicking on the test tube

Sucrose- Invertase solution Now that the enzyme has been added we will take the Sucrose-Invertase solution to our lab equipment to measure the effects of increasing temperatures on enzyme activity You will have to be quick to limit the effects of time on the actions of the enzyme with the substrate Sucrose- Invertase solution

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 2 ON 3200 3600 35o 30o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 3 ON 2800 3200 40o 35o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 4 ON 2200 2800 45o 40o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 5 ON 1400 2200 50o 45o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 6 ON 700 1400 55o 50o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 7 ON 200 700 60o 55o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 8 ON 100 200 65o 60o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 9 ON 200 100 70o 65o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 10 ON 400 200 75o 70o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

Click To Go To Next Measurement Proceed to increase the temperature of the solution in 5 degree increments by clicking on the red knob of the hot plate Next insert the temperature probe record the temperature and then turn the spectrophotometer on and record the new reading Measurement # 11 ON 800 400 80o 75o Photons Temp H C Insert Temperature Probe by clicking on it and record data Turn Spectrophotometer ON and record data Click red knob to start hot plate Click To Go To Next Measurement

ENZYME LAB At this point you should have recorded data for 11 measurements on your data table Next we will need to create a graph from the collected data which will help in interpreting the results

ENZYME LAB We will create data points on a graph in which we compare mM of product vs Temperature Use the Blue button below to view a graph where you will plot your data points You should print this graph Click to View Graph

ENZYME LAB After you have plotted all of your data points, connect all of the points by drawing a line from one data point to the next Your graph should look very similar to the graph shown from the link below Click to View Graph

Click to View Conclusion Page ENZYME LAB From the graph that you have produced create a conclusion to the experiment Did your hypothesis agree with your results Click to View Conclusion Page

Answer the following questions and include them in your lab report ENZYME LAB Print this page Answer the following questions and include them in your lab report 1) What is the optimum temperature for invertase activity? 2) Explain why temperatures lower or higher than the optimum cause decreases in invertase activity? 3) Do you think that if a different substrate like glucose was used instead of sucrose, the results would be different? Why or why not? 4) Do you think that if a different enzyme like peptase was used instead of invertase, the results would be different? Why of why not?

ENZYME LAB The following is what you should make sure that you include in your lab report 1) Hypothesis 2) Data Table 3) Graph (mM Product vs Temperature) 4) Conclusion 5) Answers to questions 1 - 4

mM Product vs Temperature 10 9 8 Mm of Product 7 6 5 4 3 2 Click to Return to Last Slide Viewed 1 30 35 40 45 50 55 60 65 70 75 80 Temperature in degrees Celsius

Click To Go To Last Viewed Slide ENZYME LAB REPORT Print this page EXPERIMENT #1 Hypothesis: Click To Go To Last Viewed Slide

Click To Go To Last Viewed Slide ENZYME LAB REPORT Print this page EXPERIMENT #1 Conclusion: Click To Go To Last Viewed Slide

Experiment #1 Data Table Click To Go To Last Viewed Slide ENZYME LAB REPORT Print this page Experiment #1 Data Table Measurement Temp Photons mM of Product 1 2 3 4 5 6 7 8 9 10 11 Click To Go To Last Viewed Slide

PHOTON TO mM of PRODUCT CONVERSION CHART To The Last Slide Viewed Photons % Density mM of Product 3600 3400 5 0.5 3200 10 1.0 3000 15 1.5 2800 20 2.0 2600 25 2.5 2400 30 3.0 2200 35 3.5 2000 40 4.0 1800 45 4.5 1600 50 5.0 1400 55 5.5 1200 60 6.0 1000 65 6.5 800 70 7.0 600 75 7.5 400 80 8.0 200 85 8.5 90 9.0 Print this page Click To Go To The Last Slide Viewed

mM Product vs Temperature Print this page 10 9 8 7 mM of Product 6 5 4 3 Click to Return to Last Slide Viewed 2 1 30 35 40 45 50 55 60 65 70 75 80 Temperature in degrees Celsius

ENZYME LAB #2 pH Influences On Enzyme Activity

ENZYME LAB #2 INDEX Lab # 2 Discussion How To Use The Spectrophotometer How To Use The pH Probe How To Use The Eye Dropper Start Of Lab # 2 Exercise Links To Charts And Graphs

ENZYME LAB The following is what you should make sure that you include in your lab report 1) Hypothesis 2) Data Table 3) Graph (mM Product vs pH) 4) Conclusion 5) Answers to questions 1 - 4

ENZYME LAB Another factor besides temperature that strongly influences enzyme activity in living cells is the pH of the environment in which the enzyme is designed to function Changes in pH can cause denaturation of enzymes thus reducing their activity

ENZYME LAB For example, in humans, a cytoplasmic protein in a skin cell is surrounded by a different fluid environment at a different pH than a membrane-bound enzyme like Invertase with an active site that projects out into the lumen of the small intestine

ENZYME LAB This exercise is designed to help you understand the effect of pH on enzyme activity by studying Invertase activity over a range of different pH values from acidic to basic conditions

ENZYME LAB To begin the experiment we will determine the pH of the substrate The substrate is the chemical material which the enzyme will be acting on

ENZYME LAB As is the case with real experiments in a biochemistry lab, enzyme should always be added to the substrate last to prevent the reaction from starting before all necessary components have been added

Click To Go To Lab Report Hypothesis ENZYME LAB The first thing you will do is to develop a hypothesis to predict the effect of changes in pH on Invertase activity Enter your hypothesis in the first line of your lab report A hypothesis is a best guess at what you think may be the results of the experiment Click To Go To Lab Report Hypothesis

ENZYME LAB The substrate used in this experiment is Sucrose Substrate concentration for sucrose for this experiment is 90 millimolar units (mM)

ENZYME LAB The enzyme used in this experiment is Invertase Invertase will react with sucrose in a biochemical reaction that results in the formation of by-products

ENZYME LAB Accumulation of by-products in the solution over time will create a change in the relative density of the solution This relative density can be measured by the use of a Spectrophotometer The use of the spectrophotometer should be familiar to you from Experiment #1 ON 4000 Photons

ENZYME LAB pH is a measure of the relative number of hydrogen and hydroxide ions present in a solution

ENZYME LAB The more hydrogen ions there are in a solution the more acidic the solution will act Less hydrogen ions and the more basic the solution will react ACID BASE H+ H+ H+ OH- OH- H+ OH- H+ OH- OH- H+ H+ H+ H+ H+ OH- H+ OH- H+ OH- H+ OH-

ENZYME LAB A device called a pH meter can be used to measure the relative number of hydrogen ions present in a solution pH of a particular solution is measured as an inverse exponential calculation of the number of hydrogen ions 7 35o pH

ENZYME LAB This means that the higher the number of hydrogen ions the lower the pH reading will be H+ H+ H+ H+ H+ pH = 6 pH = 5 H+ H+ OH- H+ OH- H+ H+ H+ H+ OH- H+ OH- H+ H+ H+ H+ H+ H+ H+ OH- H+ OH- H+ H+ H+ H+ H+ H+

ENZYME LAB Thus the lower the pH the more acidic the solution is The pH scale ranges from a low of 1 to the highest pH of 14 14 7 1

ENZYME LAB A pH measure of 7 indicates that the number of hydrogen ions and the number of hydroxide ions are equal and thus the solution will not express acidic or basic tendencies We would refer to solutions with a pH of 7 to be neutral 14 7 1 Neutral

ENZYME LAB 14 7 1 Base Neutral Acid pH measuring less than 7 would be considered acidic pH measuring higher than 7 would be considered basic Low concentrations of hydrogen ions High concentrations of hydrogen ions 14 7 1 Base Neutral Acid

ENZYME LAB HOW TO USE THE pH PROBE 7 35o pH

A pH Probe can be used to measure the acidity of the sucrose solution A pH Probe is inserted into the solution which will give measurements of hydrogen ion concentrations pH Probe 7 pH pH Meter

To measure the pH of the solution click on the pH Probe The resulting value indicated on the pH meter can now be recorded pH Probe 7 pH pH Meter

Sucrose Solution Eye Dropper HCl Acid Solution In order to change the pH of the solution to measure enzyme activity we will add a prescribed amount of acid by using an eye dropper Below you will see a bottle of acid solution and an eye dropper Sucrose Solution Eye Dropper HCl Acid Solution

HOW TO USE THE Eye Dropper ENZYME LAB HOW TO USE THE Eye Dropper

Sucrose Solution Eye Dropper HCl Acid Solution Click on the eye dropper to sample a small amount of acid A small amount of acid is now present in the eye dropper Sucrose Solution Eye Dropper HCl Acid Solution

Sucrose Solution HCl Acid Solution Click on the eye dropper again to present a single drop of acid to the sucrose solution We can now resample the sucrose solution for a change in pH Sucrose Solution HCl Acid Solution

pH Probe Spectrophotometer 4300 7 Sucrose Solution pH Meter To measure effect of pH on enzyme activity we will use a pH meter to measure acidity of the solution In order to measure the amount of product produced we will once again use a spectrophotometer as we had used in Experiment #1 ON pH Probe Spectrophotometer 4300 7 Photons pH Sucrose Solution pH Meter

Click To Go To Experiment #2 You are now ready for experiment # 2 You will be using the eye dropper to increase pH levels of the sucrose – invertase solution You will use the pH meter to measure pH and the spectrophotometer to measure enzyme activity Base Solution ON Spectrophotometer 4300 Acid Solution pH Probe Photons 7 pH Eye Dropper Sucrose Solution pH Meter Click To Go To Experiment #2

Sucrose solution Invertase The substrate solution we will use is a 90 mM sucrose solution We will be adding an enzyme to the substrate solution (Sucrose) The enzyme we will use is called INVERTASE Invertase is an enzyme which will metabolize or break down sucrose Sucrose solution Invertase When sucrose is broken down the build up of metabolic by-products causes changes in the density of the sucrose-invertase solution

In the test tube to the right is an enzyme called invertase Pour the invertase into the sucrose solution by clicking on the test tube Turn the timer on by clicking on the blue button on top of the timer and wait for 2 minutes While you are waiting you can check the pH by going to the next slide

Two Minutes have elapsed Insert the pH Probe by clicking on the probe Record the pH in your Data Table in the row for Measurement #1 When the timer has buzzed and stopped go to the next slide to test the solution for density 7 pH Two Minutes have elapsed

Turn the spectrophotometer on and record the measurement in photons in your Data Table for Measurement #1 You will need to use a conversion chart to find the amount of product produced ON 1600 7 Photons pH

Click to View Conversion Chart You can find the conversion chart by using the GREEN button below At this point you will have completed one measurement for a single pH value ON 1600 7 Photons pH Click to View Conversion Chart

HCl Acid Solution MEASUREMENT # 2 We have now acquired a fresh solution of 90 mM sucrose Add one drop of acid to the sucrose solution by clicking on the eye dropper HCl Acid Solution

MEASUREMENT # 2 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #3 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 1200 6 Photons pH Two Minutes have elapsed Click To Go To Measurement #3

Experiment #2 Data Table Click To Go To Measurement #3 ENZYME LAB At this point your Data Sheet should look like below with two measurements evaluated Experiment #2 Data Table Measurement pH Photons mM of Product 1 7 1600 5 2 6 1200 3 4 8 9 10 Click To Go To Measurement #3

Click To Go To Measurement #3 We will now proceed with measurements 3 - 10 The procedure will be to: 1) add a measured amount of acid or base to the substrate 2) add enzyme to the substrate 3) start timer for substrate enzyme action 4) measure and record pH with pH meter 5) measure and record density with the spectrophotometer ON 4300 6 Photons pH Click To Go To Measurement #3

HCl Acid Solution MEASUREMENT # 3 We have acquired a fresh solution of 90 mM sucrose Add two drops of acid to the sucrose solution by clicking on the eye dropper HCl Acid Solution

MEASUREMENT # 3 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #4 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 1000 5 Photons pH Two Minutes have elapsed Click To Go To Measurement #4

MEASUREMENT # 4 We have acquired another fresh solution of 90 mM sucrose Add four drops of acid to the sucrose solution by clicking on the eye dropper

MEASUREMENT # 4 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #5 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 1200 4 Photons pH Two Minutes have elapsed Click To Go To Measurement #5

MEASUREMENT # 5 We have acquired another fresh solution of 90 mM sucrose Add seven drops of acid to the sucrose solution by clicking on the eye dropper

MEASUREMENT # 5 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #6 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 1600 3 Photons pH Two Minutes have elapsed Click To Go To Measurement #6

MEASUREMENT # 6 We have acquired another fresh solution of 90 mM sucrose Add eleven drops of acid to the sucrose solution by clicking on the eye dropper

MEASUREMENT # 6 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #7 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 3200 2 Photons pH Two Minutes have elapsed Click To Go To Measurement #7

MEASUREMENT # 7 We have acquired another fresh solution of 90 mM sucrose Add nineteen drops of acid to the sucrose solution by clicking on the eye dropper

MEASUREMENT # 7 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #8 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 3600 1 Photons pH Two Minutes have elapsed Click To Go To Measurement #8

MEASUREMENT # 8 We have acquired another fresh solution of 90 mM sucrose Add one drop of base to the sucrose solution by clicking on the eye dropper NaOH Base

MEASUREMENT # 8 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #9 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 2400 8 Photons pH Two Minutes have elapsed Click To Go To Measurement #9

MEASUREMENT # 9 We have acquired another fresh solution of 90 mM sucrose Add five drops of base to the sucrose solution by clicking on the eye dropper NaOH Base

MEASUREMENT # 9 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Measurement #10 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 2800 9 Photons pH Two Minutes have elapsed Click To Go To Measurement #10

MEASUREMENT # 10 We have acquired another fresh solution of 90 mM sucrose Add eleven drops of base to the sucrose solution by clicking on the eye dropper NaOH Base

MEASUREMENT # 10 Add the invertase enzyme by clicking on the test tube Now click on the timer and go to the next slide

Click To Go To Next Slide MEASUREMENT # 10 Measure and record the pH of the solution by clicking on the pH probe When the timer has buzzed click on the spectrophotometer to measure and record the density ON 3600 10 Photons pH Two Minutes have elapsed You have finished collecting data for experiment #1 Click To Go To Next Slide

ENZYME LAB At this point you should have recorded data for 10 measurements on your data table Next we will need to create a graph from the collected data which will help in interpreting the results

ENZYME LAB We will create data points on a graph in which we compare mM of product vs pH Use the Blue button below to view a graph where you will plot your data points You should print this graph Click to View Graph

ENZYME LAB After you have plotted all of your data points, connect all of the points by drawing a line from one data point to the next Your graph should look very similar to the graph shown from the link below Click to View Graph

Click to View Conclusion Page ENZYME LAB From the graph that you have produced create a conclusion to the experiment Did your hypothesis agree with your results Click to View Conclusion Page

Answer the following questions and include them in your lab report ENZYME LAB Print this page Answer the following questions and include them in your lab report 1) What is the optimum pH for invertase activity? 2) Explain why pH lower or higher than the optimum cause decreases in invertase activity 3) Do you think that if a different substrate like glucose was used instead of sucrose, the results would be different? Why or why not? 4) Do you think that if a different enzyme like peptase was used instead of invertase, the results would be different? Why of why not?

ENZYME LAB The following is what you should make sure that you include in your lab report 1) Hypothesis 2) Data Table 3) Graph (mM Product vs pH) 4) Conclusion 5) Answers to questions 1 - 4 Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial 142 Previous Slide Last Slide Viewed End Show

Click To Go To Last Viewed Slide ENZYME LAB REPORT Print this page EXPERIMENT #2 Hypothesis: Click To Go To Last Viewed Slide

Click To Go To Last Viewed Slide ENZYME LAB REPORT Print this page EXPERIMENT #2 Conclusion: Click To Go To Last Viewed Slide

Experiment #2 Data Table Click To Go To Last Viewed Slide ENZYME LAB REPORT Print this page Experiment #2 Data Table Measurement pH Photons mM of Product 1 2 3 4 5 6 7 8 9 10 Click To Go To Last Viewed Slide

PHOTON TO mM of PRODUCT CONVERSION CHART To The Last Slide Viewed Photons % Density mM of Product 3600 3400 5 0.5 3200 10 1.0 3000 15 1.5 2800 20 2.0 2600 25 2.5 2400 30 3.0 2200 35 3.5 2000 40 4.0 1800 45 4.5 1600 50 5.0 1400 55 5.5 1200 60 6.0 1000 65 6.5 800 70 7.0 600 75 7.5 400 80 8.0 200 85 8.5 90 9.0 Print this page Click To Go To The Last Slide Viewed

Click To Go To Last Viewed Slide mM Product vs pH Print this page 10 9 8 Mm of Product 7 6 5 4 3 2 Click To Go To Last Viewed Slide 1 1 2 3 4 5 6 7 8 9 10 pH

Click To Go To Last Viewed Slide mM Product vs pH 10 9 8 Mm of Product 7 6 5 4 3 2 Click To Go To Last Viewed Slide 1 1 2 3 4 5 6 7 8 9 10 pH

ENZYME TUTORIAL HOW ENZYMES WORK Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial 149 Next Slide Previous Slide Last Slide Viewed End Show

HOW ENZYMES WORK Essential points about enzymes : All enzymes are proteins Each enzyme has a specific catalytic action Their normal activity depends on their environment Abnormal conditions cause reduced activity

HOW ENZYMES WORK

HOW ENZYMES WORK Enzymes are globular proteins They have an area, usually thought of as a pocket-shaped gap in the molecule, which is called the active site

HOW ENZYMES WORK Only the substrate fits into the active site The enzyme speeds up the process of conversion of substrates into products

HOW ENZYMES WORK Although the enzyme obviously joins with the substrate for a short while, the enzyme and substrate split apart afterwards, releasing the enzyme Thus the enzyme is not used up in the process, so it can continue to react if more substrate is provided

HOW ENZYMES WORK Within the normal range, changes in temperature, pH, and concentrations of substrate and enzyme affect the rate of reaction in accordance with predictable interactions between enzyme and substrate molecules

HOW ENZYMES WORK The effects of temperature may be explained on the basis of kinetic theory Increased temperature increases the speed of molecular movement and thus the chances of molecular collisions, so within a narrow range (often 0-45 °C), the rate of reaction is proportional to the temperature

HOW ENZYMES WORK It is often said that an enzyme's rate of reaction doubles for every 10° C rise in temperature However, the interaction between this positive effect of increased temperature and the negative effect of denaturation results in a different situation Enzymes may be said to have an optimum temperature for their action

HOW ENZYMES WORK Changes in the pH probably affect the attraction between the substrate and enzyme, and thus the efficiency of the conversion process Often, there is an optimum pH Near to pH 7 (neutral)

HOW ENZYMES WORK Above normal temperatures (60 °C), heat alters irreversibly the enzyme molecule This denaturation is due to molecular vibrations (caused by heat) which change the shape of the protein, altering the folding and internal cross-linkages in its polypeptide chains

HOW ENZYMES WORK These changes, especially in the region of the active site, mean that the enzyme is inactivated, even when returned to normal temperature It would be wrong to say that an enzyme is KILLED by heat, since it is only a molecule, not an organism

HOW ENZYMES WORK The higher the temperature to which the enzyme is subjected and the longer the heating is continued, the greater the proportion of damaged enzyme molecules and the result is that the conversion process becomes less and less efficient

HOW ENZYMES WORK Below normal temperatures, enzymes become less and less active, due to reductions in speed of molecular movement, but this is reversible, so enzymes work effectively when returned to normal temperature

Enzyme Tutorial is Over Return to Index HOW ENZYMES WORK Enzymes are sometimes adversely affected by other chemical substances which combine with them, either at their active site or by altering the overall shape of their molecule Many drugs, poisons and pH changes have their effect in this way Enzyme Tutorial is Over Return to Index

Extra slides

ENZYME LAB #1 INDEX Enzyme Lab Index Lab #1 Index Lab #2 Index Lab Tutorial Next Slide Previous Slide Last Slide Viewed End Show

Temperature in degrees Celsius 10 9 8 Mm of Product 7 6 5 4 3 2 1 30 35 40 45 50 55 60 65 70 75 80 Temperature in degrees Celsius

Temperature in degrees Celsius 10 9 8 Mm of Product 7 6 5 4 3 2 1 30 35 40 45 50 55 60 65 70 75 80 Temperature in degrees Celsius

Click to add 50 50o mM Product 45o 40o 35o 35o 40o 45o 50o Temperature

Spectrophotometer Temperature Probe 4300 4500 30o 35o Hot Plate ON 4300 4500 Photons 30o 35o Temp H C Hot Plate Insert Temperature Probe by clicking on it Click red knob to start hot plate Turn Spectrophotometer On

Spectrophotometer Temperature Probe 4300 4500 30o 35o Hot Plate ON 4300 4500 Photons 30o 35o Temp H C Hot Plate Insert Temperature Probe by clicking on it Click red knob to start hot plate Turn Spectrophotometer On

Temperature in degrees Celsius 10 9 8 Mm of Product 7 6 5 4 3 2 1 30 35 40 45 50 55 60 65 70 75 80 Temperature in degrees Celsius

Extra slides

mM Product vs pH 10 9 8 Mm of Product 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 pH

Click To Go To Next Slide Click on the beaker containing the solution to set it on the Hot Plate Now click on the Temperature Probe to insert it into the solution Record the measure of the temperature of the solution Temperature Probe 30o Temp H C Sucrose Solution Hot Plate Click To Go To Next Slide

Spectrophotometer pH Probe 4300 7 pH Meter ON 4300 Photons 7 pH pH Meter Insert pH Probe by clicking on it Click eye dropper to add acid Turn Spectrophotometer On