AP Biology Lab-11

7/23/2019 AP Biology Lab-11

1/22Copyright 2013 Quality Science Labs, LLC

LAB 5

Enzyme Activity

Big Idea 4: InteractionsHow do abiotic or biotic factors influence the rates of enzymatic reactions?

Please be sure you have read the

student intro packet before you do this lab.(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations SummaryPre-lab Questions

Lab Investigation 5.1: Enzyme Specificity

Lab Investigation 5.2: Enzyme Rate of Reaction Assay

and Baseline

Lab Investigation 5.3Part 1 - Determining the Effect of pH on Enzymatic Activity

Part 2 - Student Guided InquiryStudent Guided Inquiry of the Environmental Effects on EnzymeActivity



Lab 5 - Enzyme Activity

Big Idea 4: Interactions

How do abiotic and biotic factors influence the rates of enzymatic reactions?

BACKGROUND

Of the 30,000 proteins that DNA codes for, many are individual specific enzymesdesigned for driving specific reactions. Proteins are not just long chains of aminoacids. Tey are complex, 3D structures that have their folded shape determined bythe sequencing of the amino acids. One incorrect amino acid (or one nucleotidein the codon of three amino acids) in a sequence of 600 amino acids (equivalent to1800 nucleotides) can change the 3D configuration and render the protein enzymeuseless. Enzyme specificity is built into every enzyme structure.

How do enzymes work?Enzymes act as catalysts. Teylower the activationenergyneeded to drive a reaction.

Enzymes are biological catalysts. In their large folded configuration, theyhave very specifically shaped substrate binding sites. Tese binding sites makesubstrates go into the transition state (the peak of the activation energy curve).Tere is an intermediate or transitional product between reactants and finalproducts. o catalyze the reaction, several regions of the binding site must be

En

ergy

Progress of Reaction

OverallEnergy

Activation Energy

with Enzyme

Activation Energywithout Enzyme

Figure 5.0a



precisely positioned around the substrate molecules, creating active sites. Tis issimilar to a key fitting in a lock. When the enzyme attaches itself to the substratemolecule, a new temporary transitional moleculeis formed, called the enzyme-substrate complex. Within this complex, the bonds that need to be broken orformed in the reaction are in close proximity to the active site.

Because the bond between the enzymes active site and substrate causesintermolecular bond strain due to electromagnetic interactions, the bond breakingor forming of the substrate requires less activation energy. Figure 5.0c shows thebreakdown of a large molecule into two smaller products with the use of an enzyme.

All chemical reactions in biological systems require the presence of enzymes.If the 3D configuration of the protein enzyme is changed in any way, the shape ofthe binding site could be affected and the enzyme becomes nonfunctional.

How Do Biotic and Abiotic Factors Affect Enzyme Activity?pHTe optimal pH conditions are different for each enzyme. Some enzymes

function better in acid environments.Tese might be found in the stomachinvolved in digestion (like pepsin atpH of 1.5-2, a protease enzyme thatbreaks peptide bonds in proteins). Temain enzyme for this lab is lactasewhich decomposes the disaccharidesugar lactose (milk sugar) into

two monosaccharides: glucose andgalactose.(Have you noticed that sugars

end in ose and enzymes end inase?) Tere are different lactaseenzymes that function in differentpH environments. Some are moreactive in an acidic pH range from 4.0to 5.0 and some are more active in aneutral pH of 7.0.

Substrate

Enzyme

Catalytic Site

Binding Pocket

Enzyme-SubrstrateComplex

Products

Lactose + Water

Galactose

Glucose

Lactase

Figure 5.0b

Figure 5.0c



emperature emperature is an abiotic condition, and similar to pH, enzymes have optimal

temperatures at which they function best, which is usually the average temperatureof the organism. emperature can affect the enzyme by altering its shape or evenbreaking bonds. Higher temperatures can denature the enzyme protein, renderingthe enzyme irreversibly nonfunctional. Many proteins are denatured around 40 Cto 50 C, but some are still active at 70 C to 80 C, and a few even withstandboiling. If the temperature is much lower, the reaction usually goes at a slower rate.

Substrate and Enzyme ConcentrationsTe concentration of both substrates and enzymes influence the rate of enzymatic

reaction. As the enzyme concentration is increased, there is an increase in product.If there is a decrease in enzyme concentration, the rate at which the products areformed is also decreased. Enzymes can only attach to one substrate and form anESC one at a time; so the more enzymes, the faster the reaction. When saturationoccurs, all of the substrate has reacted with all of the enzymes, and the reactionstops, unless more substrate is provided.

What Other Cellular Processes Control Enzymes andAffect Enzyme Activity?

Enzymatic CompetitionEnzymatic competition occurs when there are several different enzymes

available to combine with a given substrate. Because enzymes compete with aspecific substrate site, each has fewer changes to interact with the substrate.

Negative FeedbackNegative feedback controls protein synthesis in the body. When the concentration

of products reaches a certain level, production ceases. Tis prevents too muchsynthesis of a product. Tis is one example of homeostasis at work in your body.

Activators and InhibitorsTere are many molecules other than the substrate which may interact with an

enzyme. If a molecule increases the rate of reaction it is an activator. An inhibitoris a molecule that binds to the enzyme and interferes with the formation of theESC (Enzyme-Substrate Complex), so the reaction cannot occur. Some examplesof inhibitors are detergents or organic solvents which tend to unfold the enzyme.Many inhibitors react with the enzymes side chains in or near the active sitetochange its shape or block it; such as poisonslike cyanide and curare.



How Are Enzymes Classified?Enzymatic proteins are fundamental to the survival of any living system and

are organized into a number of groups depending on their specific activities. wocommon groups are catabolic enzymes(cata-from the Greek to break down) andanabolic enzymes(a-or an-from the Greek anabole, meaning to build up).

Catalytic enzymesthat break down proteins are called proteases and are found

in many organisms. One example is bromelain, which comes from pineapples.Ever wonder why pineapple cannot be added to Jell-O? Te gelatin stays liquidand will not set. Te bromelain enzyme breaks down gelatin and is commonly usedin meat marinades to break down protein in meats. Papain is an enzyme that comesfrom papaya and is used in some teeth whiteners to break down the bacterial filmon teeth. People who are lactose-intolerant cannot digest milk sugar (lactose), butthey can take supplements containing lactase, the enzyme they are missing. All ofthese enzymes hydrolyzelarge complex molecules into their simpler components.

Hydrolysis is the process of adding water and bonds are broken, whether sugars,proteins, or nucleotides, (in the case of AP to ADP).

Anabolic enzymesare dehydration processes that remove water and build bonds.

Tey build larger molecules from smaller parts. Have you heard of the controversialanabolic steroids for muscle-building? Another example of an anabolic enzyme isAP synthase, the enzyme that stores cellular energy in AP by combining ADPand phosphate. Tese are kinase enzymes that phosphorylate or add phosphategroups. Te DNA and RNA polymerase enzymes involved in DNA replicationand RNA protein synthesis are also anabolic, building enzymes.

One more example of an anabolic enzyme is Ribulose-1,5-bisphosphatecarboxylase oxygenase, most commonly known by its nickname RuBisCo. It is anenzyme involved in the Calvin cycle that catalyzes the first major step of carbonfixation, a process by which the atoms of atmospheric carbon dioxide are madeavailable to organisms in the form of energy-rich molecules such as glucose.



Preview of Enzyme Activity Lab Investigations:Te enzymes lab investigations all use the milk sugar (lactose) enzyme lactase to

explore:

Specificity (can lactase also decompose another disaccharide besides lactose -sucrose?) in Lab Investigation 5.1

How to measure the rate of reaction of an enzyme and how to use a base lineassay if needed in Lab Investigation 5.2;

How to determine the effect of pH on enzyme activity in Part 1 of LabInvestigation 5.3; and

A student guided inquiry lab on selecting another abiotic or biotic factor thatcould affect the rate of enzyme reaction and conducting a student-designedexploration in Part 2 of Lab Investigation 5.3.



PREPARAION

Materials and Equipment are listed with each lab separately.

iming and Length of Lab ActivitiesLab Investigations 5.1 on Enzyme specificity, 5.2 on Rates of Reaction, and 5.3

on the effect of pH on enzyme activities are all using the same solutions preparedin 5.1. Lab Investigations 5.1 and 5.3 could be done in one lab period. Rates ofreaction will take longer since measurements need to be taken every five minutesfor a total of 20 minutes. Student inquiry could take two to three lab periods,depending on ExD (Experimental Design) development, conducting and collectingdata, graphing data, and discussing.

Learning objectives aligned to standards & sciencePractices (SP)

o understand the relationship between enzyme structure and function (4B1& SP 5.1).

o make some generalizations about enzymes by studying just one enzyme inparticular 4A1 & SP 6.1, 6.4).

o determine which factors can change the rate of an enzyme reaction andwhich factors that affect enzyme activity could be biologically important (2D1

& SP 4.2, 7.2).

General Safety PrecautionsBe sure to wear safety goggles when working with chemicals (pH buffers).

Since the concentration of the reactive materials in the lab investigations areenvironmentally friendly, they can be rinsed down a standard laboratory drain.Avoid rubbing your eyes or mouth when working with chemicals, even the lowestconcentrations can cause burning sensations to the eyes, lips, and mouth.



Pre-lab Questions

Hydrogen peroxide, H2

O2

, is a toxic chemical that builds up in our bodies fromthe metabolism of fats. Fortunately, our livers have a large quantity of the catalaseenzymethat breaks up the H

2O

2produced from the metabolism of fatty acids in our

livers into harmless products of water and oxygen:

2H2O

2+ Catalase enzyme2H

2O + O

2(g)

Other enzymes involved in the metabolism of certain amino acids and fattyacids (D-amino acid oxidase and acyl-CoA oxidase) make significant amounts ofhydrogen peroxide. Since hydrogen peroxide can be damaging to normal tissue,these enzymes are kept inside specialized organelles inside cells called peroxisomes.

Te peroxisomes also contain large amounts of catalase enzyme to break down thehydrogen peroxide before it can escape.

Catalase breaks down the hydrogen peroxide naturally produced by the body.Studies have found that reduction in catalase production is correlated with gray hair,suggesting that high hydrogen peroxide levels are behind gray hair. H

2O

2can also

be damaging to cells, proteins and even DNA if it is not broken down efficiently inour bodies.

If a cup of H2O

2is left standing in the open air for several days, eventually it

will spontaneously start to break up and oxygen gas will be released. However, ifwe sprinkle a little dry yeast into a cup of H

2O

2, rapid bubbling is observed. Te

catalase enzyme in yeast is acting as a catalyst and lowering the activation energy

needed to drive this reaction, releasing oxygen gas. Another quality of a catalyst isthat it does not get used up in the reaction and recycles. Te catalase enzyme candecompose over a million H

2O

2molecules in less than a second.



1. Te catalase enzyme is found in many plants and animals. Whatdo you think would happen to the protein enzyme catalase if theyeast was boiled before adding to the H

2O

2?

2. What other environmental effects do you think could denaturethe protein enzyme and cause it to be useless?



LAB Investigation 5.1:Enzyme Specificity

Te monosaccharide glucose (simple sugar) is oxidized and decomposedduring cellular respiration for energy. As bonds are broken, 36-38 AP moleculesare produced for each molecule of glucose oxidized (6-carbon simple sugar).Enzymes are needed to decomposepolysaccharides (starches, cellulose, glycogen),disaccharides (2-sugar-12carbon compounds like lactose, sucrose, maltose), andmonosaccharides (a simple 6-carbon sugar like glucose, galactose, fructose) tosupply the bodys energy needs.

Te most common disaccharides are sucrose (common table sugar)which iscomposed of two simple sugars: glucose and fructose; andlactose (milk sugar)whichis composed of two simple sugars: glucoseand galactose. In this investigation, the

enzyme lactasewill be used in an attempt to break down the disaccharides sucroseand lactose.

Materials:

Digital scale

Sucrose (table sugar), 1 gram*

Distilled water 250 mL*

Lactase tablet

Knife for cutting lactase tablet in half*

Spoons (for crushing the lactase tablet) (2)*

Whole milk, 1 mL*

24-well reaction plate

Pipets (8)

Glucose (dextrose), 2 grams

Glucose test strips (5)

Beaker plastic, 150 mL, 30 mL

Graduated cylinders, 10 mL

est tubes, plastic with caps (4)

est tube rack

Stopwatch*items not included



Preparations:

20% glucose (sometimes called dextrose a monosaccharide) solution: Add2.0 grams of glucose to 10 mL distilled water in a plastic test tube, cap the testtube and mix until it dissolves completely.

5% sucrose (table sugar a disaccharide) solution: Add 1.0 gram of sucrose to

20 mL of distilled water in a 30-mL plastic beaker, mix with a pipet until thesucrose dissolves completely; transfer to a 12-mL test tube.

Lactase (milk sugar enzyme) solution crush half a lactase tablet between twospoons over a 150-mL plastic beaker. Add 100 mL of distilled water. Mixwith a pipet until dissolved. ransfer 10 mL to a plastic test tube and cap tosave this for Lab 5.2 and Lab 5.3.

Procedures:

1. Fill in your predictions for qualitative results (using + or signs)in data able 5.1a.

2. Label and set up the following wells in a 24-well tray:

Make sure you use a clean pipet for each solution including thewater and keep the pipet with the solution for later use.

3. Obtain a glucose test strip do not touch the test pad with yourfingers. Completely immerse a glucose test strip into well Afor 1-2 seconds, run the test strip along the edge of the well toremove excess solution. (If the strip is NO blotted, the glucoseconcentration will not be accurate it may be too concentrated).

Well PlateLabel

Contents of each well:Predictions for presence of

Glucose (+ or -)est Results Concentration

of Glucose(mg/dL)

A3 drops sucrose solution +

3 drops distilled water

B 3 drops milk +3 drops distilled water

C3 drops sucrose solution +3 drops lactase solution

D3 drops milk +

3 drops lactase solution

E3 drops glucose solution +

3 drops distilled water

able 5.1a



4. Follow the directions on the glucose test strip package for timeto wait before reading the test strip. Different manufacturers teststrips vary. Te glucose test strips have specific waiting period.Do not wait any longer than the prescribed time or your colorreading will not be accurate.

5. Follow the color chart on your glucose test strip packet. Record

the glucose concentration (based on the color chart, in mg/dL)for well A in your data table.

6. Repeat Steps 2-4 for wells B, C, D, and E; using one new teststrip for each well.



Analysis:

1. What was the enzyme in this reaction? Te substrate? Te

products? Explain why testing for glucose is used to determinethe activity of the enzyme lactase.

2. What determines a persons ability to digest lactose?

3. Explain the experimental conditions for the five different wellsand the results. Which wells showed positive for glucose? Dothey match your predictions?



Lab Investigation 5.2: Enzyme Rate ofReaction Assay and Baseline

o determine the rate of an enzymatic reaction, a change in the amount ofat least one specific substrate or product is measured over time. In this lab, wewill be monitoring the change in concentration of product (glucose) from thedecomposition of lactose to the simple sugars glucose and galactose. Glucose canbe measured and quantified by color and concentration as indicated by the glucosetest strip. A control will also be run with milk and water (no enzyme) as a baseline.If there is any concentration of glucose in the control, it must be subtracted fromthe experimental results.

Materials:


Digital scale

Distilled water*

Whole milk, 1 mL*


Stopwatch

Prepared Solutions from Lab Investigation 5.1 20% glucose

5% sucrose

Lactase Enzyme Solution*item not included



Procedures

1. Set up well A with 3 drops of Milk and 3 drops of distilled water,

this well will be the control well. Set up well B with 3 drops ofmilk and 3 drops of lactase enzyme solution.

2. Immediately dip a test strip into Well A for 1-2 seconds andrecord the concentration for ime 0. (Follow same proceduresfor running the test strip along the edge of the well to removeany excess. Read color chart at time indicated on the packagingof the test strips. Record glucose concentration in your datatable. Follow the same procedure for Well B.

3. After 5 minutes, dip a new glucose test strip into Well A. Recordthe glucose concentration. Repeat every 5 minutes. Take your

last glucose strip concentration at 20 minutes. Repeat for Well B. 4. Graph your results (time on the x-axis, glucose concentration on

the y-axis).

5. Calculate the rate based on the slope of a linear line during theearly period (Y/X). Te slope of the graph during the earlyperiod is called the initial rate of the reaction. As substrateor enzyme is used up, the graph may plateau. Te rate of thereaction is the slope of the linear portion of the curve. Te ratewill be expressed as (0.055)mg/dL glucose / sec = mmoles/L ofproduct (glucose) / sec.

6. IF there was any glucose concentration detected in Well Aduring the 20 minutes, then you must use this as a baseline andsubtract it from the glucose concentrations found in Well B.

able: Presence of Glucose Indicating Lactase Activity

ime(min.)

Well A Well BRate of Reaction

(slope calculations)

0

5

10

15

20

able 5.2a



Analysis:

1. Color change was measured at different times. What time

should be used for the pH assay in Lab Investigation 5.3? Why?

2. Describe the graph line shape? Was it possible to extrapolate astraight line to determine the slope for the rate of reaction?

3. What was the result of the control? Will the experimental dataneed to be adjusted?

4. Does the rate of reaction change or is it consistently the same forevery 5 minute interval?



LAB Investigation 5.3Part 1 - Determining the Effect of pH onEnzymatic Activity

Changes in pH can alter the ionization of parts of the amino acids. Tis wouldresult in hydrogen bonding disruptions and the overall protein molecules 3D shapeand folding would change. Tis could alter the active sites and bonding abilityof the enzyme to the substrate. Te enzyme could be nonfunctional if the pH isdrastic enough to denature it. In this investigation, you will explore the effects ofvarious pH values on the functioning of the lactase enzyme.

Preparations:

pH 4.0, 7.0, and 10.0 buffers: each tube contains approximately 0.1 gram ofpowdered buffer. You need to add 10 mL of distilled water to make the buffersolution. Gently rotate the tube to mix. Te liquid buffer has a shelf life of4 weeks if stored in air tight container; the powder has a shelf life of severalyears. Using universal indicator strips, test each pH solution and record theactual pH.

Materials:

pH buffer powder in capped test tubes: pH 4, 7, 10


Whole milk*

Lactase solution (In capped test tube that you prepared in Lab 5.1)


Stopwatch

Pipets (5)

est ubes, plastic with caps (5)

Universal Indicator strips (3)*item not included



Procedures:

1. Set up the wells according to the data chart:

2. Record initial glucose concentrations at ime 0. Wait 10 minutesfor the reactions to occur and dip a new glucose test strip.

3. Follow the same procedures for completely immersing the testpad in the well for 1 second. Run the test strip along the edge ofthe well to remove excess liquid. Wait test strip instructed timefor the color to develop. Ten compare the test strip to the colorchart for concentration. Record the glucose concentration in

able 5.3a at ime 0 and at ime 10 min..

4. est the remaining wells, using a new test strip for each well andrecord the glucose concentrations at ime 0 and ime 10 min..

5. Graph the data placing glucose on the y-axis and the pH valueson the x-axis.

Wells Contents of Wells pHConcentration ofGlucose at 0 min.

mg/dL(0.055) = mmol/L

Concentration ofGlucose at 10 min. mg/dL(0.055) = mmol/L

A2 drops Buffer pH 4+ 2 drops Lactase + 2

drops Milk4

B2 drops Buffer pH 7+ 2 drops Lactase + 2

drops Milk7

C2 drops Buffer pH 10+ 2 drops Lactase + 2

drops Milk10

able 5.3a



Data Analysis:

1. What conclusions can be draws from the results?

2. What was the effect of pH on the enzyme lactase? Is this truefor all enzymes?



Part 2 - Student Guided Inquiry

Student Guided Inquiry of the Environmental Effects on Enzyme Activity

What are three or four factors that vary in the environment in which organismslive? Which of those factors do you think could affect enzyme activity?

Following are suggestions for a student guided inquiry:

Effect of Various emperatures on Enzyme Rates of Reaction

Effect of Various Enzyme and Substrate Concentrations on Rates of Reaction:Which has a greater effect on the rate of reaction changing the concentrationof enzyme or changing the concentration of substrate?

Effect of Enzymes from Different Organisms (lactase from fungi vs. lactasefrom humans)

How are organisms survival linked to enzymes?

Effect of Various Soil Salinities with different plants/seeds

Effect of Various Soil pH with different plants/seeds

Effect of light or no light in germination of seeds

Design and conduct an experiment to investigate one of the biotic or abiotic

environmental factors that influence the rate of enzymatic reactions.Step 1:

Chose your driving question to investigate.

Step 2:

Fill in the ExD (Experimental Design) form on the next page toplan your experiment.

Step 3:

Design data tables and collect data.

Step 4:

Analyze the data.

Step 5:

Summarize your conclusions.



Experimental Design (ExD) FormComplete this ExD pre-lab planning form before beginning your lab

1. Independent variable: (What is the cause agent? What are you changing?)

2. Dependent variable: (What is being measured?)

3. Lab set-up:

ExperimentalGroups

Number of Trials

4. Control: (What is the experimental group being compared to?)

5. Hypothesis: (Use an if [Independent Variable], then [Dependent Variable] format.State the cause and effect relationship between the I.V. and the D.V. It must be testable.)

6. Lab title: (e effect of ____[I.V.] ____on ____[D.V.]____.)

7. Experimental constants: (Name at least six variables NOT altered during the experiment.)

8. Sketch of experimental set up with labels:

9. Detailed procedure:


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AP Biology Lab-11

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