Biology Lab need done

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Biological Processes

Lab 4 Enzymes

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Lab 4: Enzymes

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Introduc on

Enzymes are specialized proteins that serve as biological catalysts to decrease the ac va on energy normally needed for a reac on to occur. This means the reac on rate is up to millions of mes faster than it would be without the enzyme. Most biochemical reac ons require enzymes for them to occur at fast enough rates to be useful. Typical nomenclature for enzymes follows the pa ern using the name of the substrate or the chemical reac on it catalyzes, and ends with “ ase”, e.g. catalase, amyl ase. (In other words, any me you see a word end in “ase” you know it is an enzyme).

Enzymes are extremely selec ve, and are o en described as having a “lock and key” t (Figure 1). Their shape determines which substrates they bind and interact with. The ac va on site

Figure 1: The speci city of enzymes is controlled by their lock and key t with a spe ci c substrate.

Concepts to explore:

Enzymes Selec vity Catalysts Ac va on energy Ac va on site Reac on rates

Concepts to explore:

Ac vators Inhibitors

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is the pocket where the substrate a aches and where the reac on occurs. A er the enzyme/substrate complex forms and catalysis occurs, the “new” substrate is released from the ac ve site, and the en zyme can repeat the process. Enzymes levels are not reduced or altered during the reac on. This means they are e cient and can be used repeatedly.

Enzymes determine the rate at which the reac on occurs (not how it occurs). Their ac vity is a ected by temperature, pH, enzyme and substrate concentra on, and other chemicals that may be present (such as salts, which can change the protein structure).

Varia ons in temperature and alkalinity can change the shape of the proteins, such as enzymes, which makes them inac ve (they can no longer bind to their substrate). The pH can alter charge of the pro tein, once again changing its shape and rendering them inac ve.

The concentra ons of both the enzyme and substrate determine the reac on rate (Figure 2). Remem ber that high reac on rates do not always translate into rapid me of comple on (it also depends on the amount of substrate!).

Ac vators are chemicals that bind to the ac ve site of the en zyme and help it to bind to the substrate. They are some mes called cofactors or organic coenzymes.

Inhibitors are chemicals that interfere with the binding of the substrate to the enzyme. There are two types:

Figure 2: Substrate Satura on Curve

Many drugs and poisons are en zyme inhibitors. For example, aspi rin inhibits an enzyme that leads to

in amma on.

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Compe ve (can be replaced by the substrate)

Non compe ve (not removed by the substrate)

Normal cellular processes produce toxic substances (waste) such as hydrogen peroxide and free radi cals that if not eliminated, will kill the cell. Luckily, yeast and other organisms (including humans) have an enzyme called catalase that breaks down hydrogen peroxide into oxygen and water, both harmless to cells.

Experiment 1: E ect of enzyme concentra on

Yeast cells contain catalase. The e ect of catalase can be seen when yeast is combined with hydrogen peroxide (Catalase: 2H2O2 › 2 H2O + O2). In this lab you will examine the e ects of enzyme (catalase) concentra on based on the amount of oxygen produced.

Procedure

1. Label three test tubes as 1, 2, and 3 with a permanent marker.

2. Fill each tube with 10 mL hydrogen peroxide.

3. Label three beaker as A, B, and C.

4. Add 1/2 teaspoon yeast (1 g.) to 100 mL of warm water (30 35 °C) in Beaker A. Mix well by pipe ng.

Materials

Yeast Measuring Spoon 3 Test tubes Test tube rack 3 100 mL Beakers Hydrogen peroxide 10 mL Graduated cylinder

Permanent marker Ruler String* 3 Balloons Watch* *You must provide

Figure 3: When catalsae is added to hydrogen peroxide, oxy gen is released.

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5. Make a serial dilu on of yeast solu on. To do this, measure 10 mL of the yeast solu on from Beaker A and transfer it to Beaker B. Add 90 mL warm water (30 35 °C) to Beaker B. Mix well by pipe ng.

6. Measure 10 mL of the yeast solu on from Beaker B and transfer it to Beaker C. Add 90 mL warm water (30 35 °C) to Beaker C. Mix well by pipe ng.

7. Measure and pour 5 mL from Beaker A into the rst test tube.

8. Quickly a ach a balloon to the top of the test tube so that it will ll with the oxygen produced by the enzyme reac on. It is important to execute this step quickly so that every bit of gas pro duced will be captured.

9. Swirl each tube to mix, and wait one minute.

10. A er one minute has passed, wrap the string around the center of the balloon to measure the circumference. Measure the length of string with a ruler. Record the length in Table 1 below.

11. Repeat step 10 a er two more minutes have passed (three minutes total from the start of the reac on); and again a er two more minutes have passed ( ve minutes total from the start of the reac on). Record all data in Table 1.

12. If the reac on has not nished, con nue to monitor how long it takes for the reac on to com plete, and measure the nal balloon circumference.

13. Repeat steps 7 12 for the remaining test tubes (use beaker B for test tube 2 and beaker C for test tube 3).

Table 1: E ect of enzyme concentra on on the produc on of gas

Ques ons

1. What is the enzyme in this experiment? What is the substrate?

Tube Amount of yeast

Circumference (cm) A er 1 minute

1 0.05 g

2 0.005 g

3 0.0005 g

Circumference (cm) A er 3 minutes

Circumference (cm) A er 5 minutes

Time Required to Complete

Final Circumference (cm)

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2. Did you no ce a di erence in the rate of reac on in the tubes with di erent concentra ons of enzymes? Why or why not?

3. What was the e ect of using less enzyme on your experiment?

4. Do you expect more enzyme ac vity if the substrate concentra on is increased or decreased? Draw a graph to illustrate this rela onship.

5. Hydrogen peroxide is toxic to cells, yet is a common byproduct of the reac ons that occur in side the body. How can this compound be changed to become non toxic (Hint: Look at the chemical formula of hydrogen peroxide)?

Experiment 2: E ect of temperature on enzyme ac vity

This experiment looks at the e ect of temperature on enzyme ac vity.

Procedure

1. With a permanent marker, label the test tubes as 1, 2, 3, and 4. Place the test tubes in the test tube rack for support.

Materials

Yeast Measuring spoon 4 Test tubes 40 mL Hydrogen peroxide, H2O2 10 mL Graduated cylinder 4 Balloons 2 Water bath containers* Pot for boiling water* Stove top*

Hot Pad* 4 Microwave safe cups* Permanent marker Test tube rack Ruler String* Watch* Thermometer *You must provide

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2. Use the 10 mL graduated cylinder to measure and pour 10 mL of hydrogen peroxide into each test tube.

3. Fill a pot with approximately 2 3 inches of water and place it on the stove (turned to medium high se ng). The water should come to a boil (approximately 100 °C).

4. While the water is hea ng, place each tube in separate, microwave safe cups (or beakers).

5. Gather two containers that can be used as hot water baths. Each container should be wide enough to t the cup with the test tube in it.

6. Pour 2 3 cups of water into a microwave safe container and heat the water un l it has reached approximately 85 °C (use the thermometer to monitor this). Pour this water into the rst hot water bath.

7. Using a hot pad, pour the boiling water from the stove into the second hot water bath.

8. Immediate place the cup holding test tube 1 into the boiling hot water bath and the cup hold ing tube 2 into the hot (but not boiling) water bath. Keep test tube 3 at room temperature, and place the cup with test tube 4 in the refrigerator. You may need to add weight (e.g., coins, mar bles, rocks, etc.) to the cups going into the water baths to keep them from pping over into the water.

9. Record the ini al temperatures of each condi on in the table below. Let tubes sit for approxi mately 15 minutes, and record the nal temperature.

10. A er the elapsed me, remove the tubes from their respec ve environments.

11. Add 1/4 tsp. of yeast to the refrigerated test tube.

12. Quickly a ach a balloon to the top of the test tube so that it lls with the oxygen produced from the enzyme reac on occurring in the tube. It is important to execute this step quickly so that every bit of gas produced is captured.

13. Swirl the tube to mix, and wait one minute.

14. A er one minute has passed, wrap the string around the center of the balloon to measure the circumference. Measure the length of string with a ruler. Record the length in Table 2 below.

15. Repeat step 14 a er two more minutes have passed (three minutes total from the start of the reac on); and again a er two more minutes have passed ( ve minutes total from the start of the reac on). Record all data in Table 2.

16. If the reac on has not nished, con nue to monitor how long it takes for the reac on to com plete, and measure the nal balloon circumference.

17. Repeat steps 11 16 for the remaining three test tubes.

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Table 2: E ect of temperature on the produc on of gas

Ques ons

1. What is the enzyme in this experiment? What is the substrate?

2. How does temperature a ect enzyme func on?

3. Do plants and animals have an enzyme that breaks down hydrogen peroxide? How could you

test this?

4. How did the boiling water a ect the overall reac on?

5. How can enzyme ac vity be increased?

6. Design an experiment to determine the op mal temperature for enzyme func on, complete

with controls. Where would you nd the enzymes for this experiment? What substrate would

you use?

7. Draw a graph of balloon diameter vs. temperature. What is the correla on?

Tube Ini al

Temp. °C

Circumference (cm) A er 1 minute

Refrigerator

Room temperature

Hot water (~85 °C)

Boiling Water (~100 °C)

Final Temp. °C

Circumference (cm) A er 3 minutes

Circumference (cm) A er 5 minutes

Final Circumference

(cm)

Time Required to Complete

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