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Lab 2: Enzyme Activity LAB GOALS: 1. To study protein structure and function 2. To examine conditions that alter enzymatic function by altering structure (denaturation) 3. To design a meaningful experiment 4. To learn to use a digital micropipette

INTRODUCTION TO LAB Today we are delving into the inner workings of the cell in order to examine one of the most important aspects of cellular function. This lab will help you synthesize information about protein structure, carbohydrate structure, and cellular reactions. The thousands of chemical reactions occurring in a cell each second are not random events but are highly controlled by biological catalysts called enzymes. Enzymes are a type of protein. All enzymes are proteins but not all proteins are enzymes. All proteins have a unique shape due to their individual amino acid sequences. The shape of an enzyme, especially in its active site, determines its catalytic effects. The active site of each type of enzyme will bind only with certain molecules. A molecule that binds with an enzyme and undergoes chemical modification is called the substrate of that enzyme.

The binding between enzyme and substrate consists of weak, non-covalent chemical bonds, forming an enzyme-substrate complex that exists for less than a millisecond and serves to stabilize the transition state. During this time, the substrate is converted to a new type of molecule called the product of the reaction. The product then leaves the enzyme's active site. The enzyme, unchanged by the reaction, is free to enter the catalytic cycle again, provided there are other substrate molecules available. Individual enzyme molecules may complete the catalytic cycle several thousand times per second. Therefore, a small amount of enzyme can convert large quantities of substrate to product. Eventually enzymes wear out just as any other protein would; they break apart and lose the capacity for catalytic activity. When no enzyme is present, the chemical reaction catalyzed by the enzyme usually will not occur to any appreciable extent.

ENZYME STRUCTURE This topic will be covered in lecture and is also part of your assigned reading. Before coming to lab this week, review protein/enzyme structure (sections 3.2–3.4) in your textbook.

PROTEIN/ENZYME DENATURATION An enzyme’s function is directly related to its overall shape (also called tertiary structure or conformation) since it is the tertiary structure that determines the shape of the enzyme’s active site. The tertiary structure of an enzyme is determined by interactions between the R-groups of the amino acids that compose the enzyme. These interactions include non-covalent interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions as well as covalent disulfide bonds. Refer to Fig 3.11 in your textbook to review these types of interactions. What do you predict would happen to the overall shape of an enzyme if any of these interactions were disrupted? Would the enzyme retain its characteristic conformation? The answer is, of course, no. Disrupting the interactions that stabilize the tertiary structure of an enzyme will cause the enzyme to lose its original shape and become denatured. Denatured enzymes lose their ability to catalyze

PLEASE complete your Pre-lab

before the start of the first TUESDAY lab

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reactions. What types of conditions might cause enzyme denaturation? Conditions that alter the hydrogen bonds, ionic bonds, disulfide bonds, and hydrophobic interactions that stabilize tertiary structure include: temperature, pH, and salt concentration. Temperature can directly affect the rate of an enzymatic reaction. Keep in mind that all chemical reactions are affected by temperature, according to the laws of thermodynamics. An increase in temperature results in an increase in molecular motion. This makes collisions between the enzyme and substrate more likely, and therefore causes the reaction to occur at a faster rate. The opposite is true as well: as the temperature decreases, so does molecular motion. This makes collisions between the enzyme and substrate less likely, and therefore causes the reaction to occur at a slower rate. At high temperatures, the molecular motion is so great that many of the interactions that stabilize the enzyme’s tertiary structure are disrupted and the enzyme is denatured. Extreme pH levels can also denature enzymes. Cellular environments typically have a near-neutral pH, thus most enzymes function best around pH ~ 7. Many biologically relevant molecules, such as metabolic byproducts, can act as acids or bases. Recall that in an aqueous solution, acidic molecules release H+ ions, lowering the pH, and basic molecules bind to and remove H+ ions, increasing the pH. Some interactions that stabilize the tertiary structure of an enzyme, such as hydrogen bonds and ionic bonds, can be disrupted by being in a very acidic or basic environment. High concentration of ions, such as salts, can also denature enzymes. High ion concentrations interfere with hydrogen bonds and ionic bonds, causing the protein to lose its characteristic conformation.

MILK SUGAR DIGESTION Most humans consume less and less milk as they age. It is mostly the peoples of Northern European descent and a few nomadic African tribes that practice the strange custom of consuming milk in adulthood. In fact, many adults of the world lack the enzyme that is required to break down lactose, the disaccharide that makes up more than half of the calories found in cow’s milk. After mammals are weaned, they typically do not encounter lactose in their diet. Besides milk, the only other significant sources of lactose are forsythia flowers and a few tropical shrubs. It only makes sense that the body would stop making an enzyme for which it has no use. When large amounts of lactose are consumed by people who no longer have the enzyme to break it down, the sugar passes into the lower intestine, where bacteria can use it as food. Much to the chagrin of these folks, this causes gas, water retention, and diarrhea.

The enzyme in cells that breaks down lactose into its two monosaccharide components, galactose and glucose, is called ß-galactosidase (in bacteria) or lactase (in humans). In this lab we will study a product called LacteezeTM, which is a trademark name for ß-galactosidase produced by a company in Canada. We will also study LactaidTM milk, which is a trademark name for milk that has been treated with ß-galactosidase. According to the manufacturers of these products, people who are lactose intolerant can treat their milk with Lacteeze, or drink Lactaid milk, and enjoy it without gastric distress. We will check out the claims that the enzyme in Lacteeze has the advertised catalytic properties and determine what types of environmental conditions can denature this enzyme.

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THE ASSAY Since we cannot directly see glucose or galactose molecules, the products of the breakdown of lactose, we need an assay. An assay can be used to determine whether or not the enzyme is present and functioning properly. One assay could be to taste the products of our reactions. Lactose is one of the least sweet-tasting sugars, yet galactose and glucose are very sweet. Therefore, by tasting the reactions, and rating them for sweetness, we could tell whether or not the reaction has occurred. However, we will not use this assay for two reasons. First, sweetness is a subjective measure. Second, it is never safe to taste anything handled in a laboratory! Instead, an assay has been developed to test ß-galactosidase activity using a different substrate (the molecule that is changed by the enzyme). Instead of lactose, we will use a compound called ONPG. ONPG is a compound consisting of a ß-galactose molecule connected with a ß-linkage to ONP (ortho-nitrophenol, see below). In using this molecule, we are taking advantage of the fact that the enzyme ß-galactosidase recognizes only the ß-galactose portion of the lactose molecule. Therefore, ß-galactosidase will also hydrolyze a variety of compounds containing a ß-galactose connected to something else. Here, that something else is ONP. The other cool aspect of this assay is that ONPG is a colorless compound, while ONP by itself is yellow! Therefore we can observe ß-galactosidase activity in solution by watching yellow ONP product being produced. We could even quantify enzyme activity by measuring the total amount of yellow produced using a spectrophotometer. However, for our purposes today we will use our eyes to provide a qualitative measure of yellowness. In this assay, ONPG is considered an artificial substrate of the enzyme – it is not a substance found in nature, but one created in a laboratory.

Colorless Yellow

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PART I: Pipetting DIGITAL MICROPIPETTES Researchers commonly use digital micropipettes today. These devices are accurate and easy to use. Very small volumes can be measured with them.

Digital micropipettes are made to handle different volume ranges. The most common ones used for small volumes are the P-20, P-200, and P-1000 micropipettes. The number after the “P” refers to the MAXIMUM volume in microliters (µl) that can be dispensed by the pipette.

P-20 2 µl-20 µl range P-200 20 µl-200 µl range P-1000 100 µl-1000 µl range The piston tops of the P-1000, P-200, and P-20 micropipettes. Used with permission; Marcia Bhide, Edu-graphics.com. Important to note: These micropipettes accurate in the ranges they are designed for. However, they will break and/or lose their calibration if you attempt to set them to a volume outside of their operating range. Never move the setting knob of a micrometer outside of its range. The volume on a micropipette is set by turning a knob near the top of the handle and watching the dial change to the correct setting. The value shown on the dial depends on the range of the micropipette. All the values are in microliters (µl). On the P-20, the last digit is 1/10 of a microliter (an invisible decimal point comes just above it). The P-200 and P-1000 read microliters to the right of the decimal point (ones, tens, and 100s). Never turn the dial past the range of the pipette.

A micropipette has a plunger with a flat knob at the top for withdrawing and dispensing liquids. In pressing down this knob, you will notice it has two stops; the second stop is felt after you push down harder after hitting the first stop. Push down to the first stop before entering a solution to be transferred. Push down to the second stop to dispense liquids into another tube (this adds an extra puff of air to get it all out).

This micropipette is a precisely-tuned instrument. It is useful to note that the volume distributed may change slightly if the micropipette is not straight up and down while in use, and if the dial has been twisted past it’s normal range. Never hold or place the micropipette upside-down to rest (where fluid can flow up the barrel and be inadvertently dispensed later, messing up your and subsequent users experiments) or touching the end of the un-tipped barrel to any surface (where it could become contaminated or bent). Lastly, modern tips go onto the barrel very easily; be gentle.

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Directions for using digital micropipettes (Your TA will review these with you in lab):

1. Set your volume by twisting the knob to so that the desired volume appears in the window. Be careful not to exceed the range!

2. Put a tip on the micropipette. This tip is disposable and is often changed to prevent cross-contamination. Never use your micropipette without a tip attached—solution sucked up into the micropipette will cause miscalibration, corrosion of its internal parts, and contamination of solutions that used subsequently.

3. Depress the flat circular knob on the top of the micropipette to the first stop.

4. Put the tip of the pipette in the liquid. 5. SLOWLY release the pressure on the flat knob.

6. After the appropriate volume has entered the pipette, remove the tip from the liquid.

7. Put the tip into the vessel to which you are transferring the liquid. When dispensing small volumes, e.g., from a P20, place the tip on the wall of the vessel near the bottom, or the top of the solution that is already present. This will ensure that all of the liquid being dispensed will be mixed into your reaction.

8. Slowly depress the flat knob past the first stop until you can push it down no further.

9. Remove the pipette from the solution. 10. Change tip before pipetting another solution, unless you are pipetting the same solution into dry

vessels.

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PART II: Exploring The ß-Galactosidase Reaction In order to form a complete reaction, three essential components are necessary: • Enzyme: ß-galactosidase (Lacteeze); measured with the P20

• Substrate: ONPG; measured with the P1000

• Reaction stop: Na2CO3 (sodium carbonate); measured with the 10 ml pipette and syringe This first set of experiments will help you understand how to design and use positive and negative controls.

1. Place 3 test tubes in the white rack and label them 1-3.

2. To the first tube, add 1 ml of ONPG + 10 µl of ß-galactosidase. Mix the contents of the tube by “flicking” with the tip of your finger (see figure below). Wait one minute and add 3 ml of Na2CO3. Mix again.

3. To the second tube, add 1 ml of ONPG + 3 ml of Na2CO3. Mix. Wait one minute and add 10 µl of ß-galactosidase. Mix again.

4. To the third tube add 3 ml of Na2CO3+ 10 µl of ß-galactosidase. Mix. Wait one minute and add 1 ml of ONPG. Mix.

5. Compare the three tubes. The intensity of yellow in the tubes can be represented using a number scale, with a rating of ‘5’ being very yellow, a rating of ‘3’ being medium yellow, and rating of ‘1’ having no yellow coloring, etc.

6. Assign a numerical rating to each of your tubes.

Note: This is an excellent example of shorthand notation. The sequence of reagents and their order of addition to follow enzyme activity.

Record your results and answer the questions on the following page.

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Names _____________________________________________________ Lab Section ________ Part II: Analysis Of The ß-Galactosidase Reaction Draw a bar graph to compare the relative amounts of yellow coloring in each tube.

5 4 3 2 1 ONPG

+ ß-galactosidase

ONPG +

Na2CO3

Na2CO3 +

ß-galactosidase 1 min 1 min 1 min Na2CO3 ß-galactosidase ONPG

1. What is the function of each of the three elements in the reaction? ONPG? ß-galactosidase (Lacteeze)? Na2CO3? 2. Did any of your tubes turn very yellow? Did any of your tubes stay colorless? The intensity of

yellow is directly correlated to the amount of ß-galactosidase activity in each tube. What did this exercise tell you about the ß-galactosidase reaction?

Amount of yellow

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Now that you have an understanding of what is happening during the ß-galactosidase reaction, let’s investigate how changing environmental conditions affect the reaction. Remember that ß-galactosidase is an enzyme and that enzymes generally have particular conditions under which they work best (self-test: why are enzymes picky about environmental conditions?). Enzymes are sensitive to pH, temperature, the concentration of dissolved salts, and the concentration of other dissolved solutes (which can stimulate or inhibit the enzyme). What do you think will happen to the activity level of ß-galactosidase if these environmental conditions are altered?

Your TA will assign your group an environmental variable to test. Working with your team members, decide what question you will investigate. Next, write a hypothesis, determine the experimental design and carry out an experiment to test your hypothesis. Make sure you include appropriate controls in your experiment! Use the worksheet on the following pages to record your question, hypothesis, and plan your experimental design, etc. **PRIOR TO CARRYING OUT YOUR EXPERIMENT, DISCUSS YOUR HYPOTHESIS AND PROPOSED EXPERIMENTAL DESIGN WITH YOUR TA**

After your team has completed your experiment, share your results with the other teams by giving a brief presentation. Since you will ultimately be responsible for understanding the experimental design and the data from all of the experiments performed by the class, be sure to copy down the results during other group’s presentations. There is space for you to record the experimental data in “Summary of All Experiments”. After all groups have shared their findings, your TA will summarize the findings and will address any results that are typical and those that might be anomalous. Clean Up Instructions: • Tubes: Empty liquid waste into the large jar marked “ONPG Waste”.

(We dispose of colored chemicals differently to avoid unnecessary pollution.) Be sure to rinse out the tubes thoroughly! Partially rinsed tubes can cause errors in future labs. Clean tubes and place in white basket, top down, to dry.

• Pipette tips: Place in the ‘lab glass’ cardboard box in the back of the room.

• ONPG: Return to the refrigerator in Room 143.

• Na2CO3: Make sure the cap is on. Leave on benchtop.

• Benches: Wipe down with paper towel/ disinfectant, remove all sodium bicarbonate buildup.

• Experiment Materials: Neatly return all materials to the plastic coffin (this includes pipettes, etc).

• Wash your hands!

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Names_____________________________________________________ Lab section________ 1. What question are you investigating?

2. State your hypothesis:

3. Experimental design. Write the details of your experiment below in appropriate and clear

shorthand. Address the following in your shorthand notation:

(Note: It may be helpful to draw reaction tubes and describe their contents/reaction conditions)

a. How many reaction tubes do you need to set up to answer this question?

b. How should the reactions be set up? (Hint: Do you need to get the enzyme into reaction conditions before adding the substrate?)

c. What will you add to each tube? How much? What order?

d. How long will the reaction run?

e. You must also have a positive control for your experiment to verify that your enzyme is functional and to calibrate the highest level of yellowness (You may not use the most yellow tube from the previous exercise!).

4. Prediction under hypothesis (include reference to specific measurements):

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5. Summary of data/results: (Hint: Draw out a quick sketch of the expected graph of data before the experiment. This is always a good practice, as it helps with later interpretation of the data.)

6. Conclusions:

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SUMMARY OF ALL EXPERIMENTS You are responsible for understanding the experiments and results of all experiments performed in lab today. The space is provided for you to record this information during the presentations. 1. pH

Example of experimental set up: Control: Conclusion: 2. Temperature Example of experimental set up: Control: Conclusion:

5 4 3 2 1

5 4 3 2 1

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3. Salts

Example of experimental set up: Control: Conclusion: 4. Sugars

Example of experimental set up: Control: Conclusion:

5 4 3 2 1

5 4 3 2 1

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5. Regular Milk

Example of experimental set up: Control: Conclusion: 6. Lactaid Milk Example of experimental set up: Control: Conclusion:

5 4 3 2 1

5 4 3 2 1

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DISCUSSION QUESTIONS Names _____________________________________________ Lab Section ________ 1. In this lab, we tested whether enzyme activity was affected by changes in the following

conditions. First, state in complete sentences the conclusion was reached by changing each variable. Then, provide a molecular explanation for each conclusion. Be specific here -– your explanation should show that you understand how enzymes work at the molecular level.

a) pH:

b) Temperature:

c) Salt: 2. In the alternate sugar experiment, we used the following sugars: raffinose, lactose, glucose,

galactose, and sucrose. Examine the structures of these sugars. Your experimental data probably shows that these sugars can be classified into two groups: sugars that do compete with ONPG and sugars that do not compete. Which sugar is in which group?

3. What is the difference between the two groups? Here again, use your answers to demonstrate

your knowledge of how enzymes work.

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Below is an abstract taken from a review written by D. Swallow, an expert in the human genetics of lactose persistence and intolerance. The review appeared in the Annual Review of Genetics in 2003.

“Lactase activity is high and vital during infancy, but in most mammals, including most humans, lactase activity declines after the weaning phase. In other healthy humans, lactase activity persists at a high level throughout adult life, enabling them to digest lactose as adults. This dominantly inherited genetic trait is known as lactase persistence. The distribution of these different lactase phenotypes in human populations is highly variable and is controlled by a genetic difference mapping to the lactase gene. A nucleotide change has been identified that occurs frequently in Northern Europeans, where lactase persistence is frequent. “ Discuss the meaning of this abstract with your lab partners. If you are confused about any of the terms, you can ask your TA to help you define them. The molecular basis of genetics will be a focus for the remainder of Bio200.