Experiment Lab 10 on Enzymes | Lab Reports Biology

Experiment 10 – Enzymes

Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like

all catalysts, speed up reactions without being used up themselves. They do this by

lowering the activation energy of a reaction. All biochemical reactions are catalyzed by

enzymes. Since enzymes are proteins, they can be denatured in a variety of ways, so they

are most active under mild conditions. Most enzymes have optimum activity at a neutral

pH and at body temperature.

Enzymes are also very specific – they only act on one substrate or one class of

related substrate molecules. The reason for this is that the active site of the enzyme is

complementary to the shape and polarity of the substrate. Typically, only one kind of

substrate will “fit” into the active site.

In this experiment, we will work with the enzyme amylase. This enzyme is

responsible for hydrolyzing starch. In the presence of amylase, a sample of starch will be

hydrolyzed to shorter polysaccharides, dextrins, maltose, and glucose. The extent of the

hydrolysis depends on how long it is allowed to react – if the starch is hydrolyzed

completely, the resulting product is glucose.

You will test for the presence or absence of starch in the solutions using iodine

(I2). Iodine forms a blue to black complex with starch, but does not react with glucose. If

iodine is added to a glucose solution, the only color seen is the red or yellow color of the

iodine. Therefore, the faster the blue color of starch is lost, the faster the enzyme amylase

is working. If the amylase is inactivated, it can no longer hydrolyze starch, so the blue

color of the starch-iodine complex will persist.

You will also test for the presence of glucose in the samples using Benedict’s

reagent. When a blue solution of Benedict’s reagent is added to a glucose solution, the

color will change to green (at low glucose concentrations) or reddish-orange (at higher

glucose concentrations). Starch will not react with Benedict’s reagent, so the solution will

remain blue.

Effect of Enzyme Concentration

During catalysis, the first step is the substrate (S) binding to the enzyme (E),

giving an enzyme-substrate complex (ES). This is an equilibrium reaction, and will be

favored by a high concentration of enzyme and/or substrate. After the substrate is bound,

the reaction takes place, and then the product is released.

E + S ⇔ ES à E + P

Effect of Temperature

All reactions are faster at a higher temperature. However, enzyme-catalyzed

reactions become slower or stop if the temperature becomes too high, because enzymes

become denatured at high temperatures. Therefore, enzymes have an optimum

temperature that corresponds to maximum activity. (At higher or lower temperatures, the

activity of the enzyme is lower.) The optimum temperature is usually around body

temperature (37°C).

Effect of pH

Each enzyme has an optimum pH. Above or below an enzyme’s optimum pH, its

activity is lower. The optimum pH of a particular enzyme corresponds to the pH of its

natural environment. For many enzymes, this corresponds to pH values of around 7. For

pepsin, which is active in the stomach, the optimum pH is 2 (the pH of the stomach).

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Experiment 10 – Enzymes

Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all catalysts, speed up reactions without being used up themselves. They do this by lowering the activation energy of a reaction. All biochemical reactions are catalyzed by enzymes. Since enzymes are proteins, they can be denatured in a variety of ways, so they are most active under mild conditions. Most enzymes have optimum activity at a neutral pH and at body temperature. Enzymes are also very specific – they only act on one substrate or one class of related substrate molecules. The reason for this is that the active site of the enzyme is complementary to the shape and polarity of the substrate. Typically, only one kind of substrate will “fit” into the active site. In this experiment, we will work with the enzyme amylase. This enzyme is responsible for hydrolyzing starch. In the presence of amylase, a sample of starch will be hydrolyzed to shorter polysaccharides, dextrins, maltose, and glucose. The extent of the hydrolysis depends on how long it is allowed to react – if the starch is hydrolyzed completely, the resulting product is glucose. You will test for the presence or absence of starch in the solutions using iodine (I 2 ). Iodine forms a blue to black complex with starch, but does not react with glucose. If iodine is added to a glucose solution, the only color seen is the red or yellow color of the iodine. Therefore, the faster the blue color of starch is lost, the faster the enzyme amylase is working. If the amylase is inactivated, it can no longer hydrolyze starch, so the blue color of the starch-iodine complex will persist. You will also test for the presence of glucose in the samples using Benedict’s reagent. When a blue solution of Benedict’s reagent is added to a glucose solution, the color will change to green (at low glucose concentrations) or reddish-orange (at higher glucose concentrations). Starch will not react with Benedict’s reagent, so the solution will remain blue. Effect of Enzyme Concentration During catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favored by a high concentration of enzyme and/or substrate. After the substrate is bound, the reaction takes place, and then the product is released. E + S ⇔ ES à E + P Effect of Temperature All reactions are faster at a higher temperature. However, enzyme-catalyzed reactions become slower or stop if the temperature becomes too high, because enzymes become denatured at high temperatures. Therefore, enzymes have an optimum temperature that corresponds to maximum activity. (At higher or lower temperatures, the activity of the enzyme is lower.) The optimum temperature is usually around body temperature (37°C). Effect of pH Each enzyme has an optimum pH. Above or below an enzyme’s optimum pH, its activity is lower. The optimum pH of a particular enzyme corresponds to the pH of its natural environment. For many enzymes, this corresponds to pH values of around 7. For pepsin, which is active in the stomach, the optimum pH is 2 (the pH of the stomach).

Trypsin, which is active in the small intestine, has an optimum pH of 8 that matches the pH of the small intestine. Effect of Inhibitors Inhibitors are substances that slow down or stop enzymes. Competitive inhibitors are molecules that are very similar to the substrate, so they can bind to the enzyme but cannot react. They compete with the substrate for the active site of the enzyme. Noncompetitive inhibitors are molecules that are not similar to the substrate and therefore do not bind to the active site. They do, however, bind to a different location on the enzyme and change the shape of the active site so that the substrate can no longer bind. Irreversible inhibitors form covalent bonds to the enzyme and therefore cannot be removed. Safety Precautions:

Wear your safety goggles. Waste Disposal:
All waste must be placed in the inorganic waste containers (which have a blue label) in one of the fume hoods.

Procedure

Important: read the entire procedure and make sure you understand it before you start this experiment. Before you start each part of the experiment, make sure you will have enough time to complete it. Advance planning is very important for this experiment. Preparation: Constant temperature water baths will be needed for this experiment. The laboratory has a large water bath that can be set to 37°C, and everyone will use this water bath. You will also need a low temperature and a high temperature water bath, and you can make your own. For the low temperature bath, use a 250-mL or 400-mL beaker, fill it about halfway with tap water, and add some ice to the water. The temperature of this bath when it comes to constant temperature should be between 0 and 5°C. To make the high temperature water bath, fill a 250-mL or 400-mL beaker about two-thirds full and heat it until it boils. (You can heat it on a hotplate or on a Bunsen burner.) When it boils, you can reduce the heat, but keep it boiling. The temperature of this bath should be close to 100°C. You will need some 1% starch solution for each part of the experiment. Shake up the bottle of starch, and collect about 50 mL of the solution in a small beaker. You will also need some of the iodine reagent for each part of the experiment. If dropper bottles of the iodine reagent are available, obtain one of them. If there are no dropper bottles, collect a small amount of this reagent (about 3-5 mL) in a small beaker,

back in the 37°C water bath. Record the time at which you added enzyme. Go on immediately to the next step.

Transfer four drops of each reaction mixture (tubes 1-4) using a separate clean dropper for each to separate wells on a spot plate. (Keep the test tubes in the water bath.) Add one drop of the iodine reagent to each. Record your observations. Use the visual color reference table to assess the enzyme activity. After you have recorded your observations, rinse off the spot plate.
Repeat step 3 after the mixtures have had five minutes of reaction time.
Repeat step 3 after the mixtures have had ten minutes of reaction time.
In the test tubes where hydrolysis occurred, the presence of glucose can be confirmed with Benedict’s reagent. Add 3 mL of Benedict’s reagent to each test tube and put them in the boiling water bath for 3-4 minutes. The appearance of a green to orange or red precipitate indicates the presence of glucose. If the solution remains blue with no solid, then no glucose is present and no hydrolysis of the starch has taken place.
Make a graph of the enzyme activity vs. the amount of amylase solution using your results at 10 minutes. Part 2: Effect of Temperature
Place 4 mL of 1 % starch solution in each of three clean test tubes. Place 4 mL of amylase solution in 3 separate clean test tubes (so you will have a total of six test tubes: 3 containing starch and 3 containing enzyme).
Place a starch tube and an amylase tube in the 37°C water bath. Place one tube of each in an ice-water bath, and one of each tube in a boiling water bath. Keep the tubes in their baths for 10 minutes to allow them to reach the temperature of their baths.
Read and record the temperature of the ice-water bath. Pour the amylase solution into the starch solution, mix, and put the tube back into the bath. Record the time.
Repeat step 10 for the 37°C bath.
Repeat step 10 for the boiling water bath.
For each mixture, after 10 minutes of reaction have elapsed, transfer 4 drops of the mixture to a spot plate. Add 1 drop of iodine reagent to each sample. Record the color and activity level of the enzyme in each case.
Graph the enzyme activity level vs. temperature. Part 3: Effect of pH
Label four test tubes pH 2, 4, 7, and 10. In each tube, place 4 mL of the appropriate buffer (with the corresponding pH). Add 4 mL of amylase solution to each of the tubes. Get 4 additional clean test tubes, and put 4 mL of 1% starch solution in each tube. Place all 8 of these tubes in the 37°C water bath for about 5 minutes to allow the temperature to equilibrate. Rinse out four separate droppers.
Pour the contents of each starch test tube into a different amylase-buffer test tube. Mix them and return the tubes to the 37°C water bath.
After 15 minutes have elapsed, transfer 4 drops of each reaction mixture (using clean droppers) to a spot plate. Add 1 drop of iodine reagent to each. Record your observations and determine the enzyme activity level for each mixture. Rinse off the spot plate with deionized water.
Make a graph of the enzyme activity level vs. pH. Part 4: Effect of Inhibitors

Place 4 mL of amylase solution in each of three test tubes. To the first tube, add 10 drops of 1 % NaCl solution. Add 10 drops of 95 % ethanol to the second test tube. Add 10 drops of either AgNO 3 or Pb(NO 3 ) 2 solution to the third test tube.
In each of three separate clean test tubes, place 4 mL of 1 % starch.
Place all six test tubes in the 37°C water bath for 5 minutes. When the 5 minutes is up, pour a tube of starch solution into each of the enzyme-inhibitor mixures. Mix each tube and return the tubes to the water bath for 15 minutes. Meanwhile, rinse out three droppers.
After 15 minutes, transfer 4 drops of each mixture (using a clean dropper each time) to a spot plate. Add 1 drop of iodine reagent to each sample. Record your observations and the enzyme activity level in each case.

Questions

What is the substrate of the enzyme α–amylase?
What are the products of the hydrolysis of the substrate?
In part 1, at which enzyme concentration was starch hydrolyzed the fastest? Slowest?
Describe the effect of enzyme concentration on enzyme activity.
In part 2, what was the optimum temperature for amylase?
Why did the enzyme activity differ at 0°C and at 100°C?
How is the activity of amylase affected by a low pH? By a high pH? Explain.
What was the optimum pH for amylase?
Pepsin hydrolyzes proteins in the stomach. The pH in the stomach is 2, and the optimum pH for pepsin is 2. What do you think would happen to the activity of pepsin when it reaches the small intestine, where the pH is 8? Explain why.
In part 4, in which reaction mixtures did hydrolysis of starch occur?
What substances added to the mixtures were inhibitors?
Explain how an organic solvent like hexane could inhibit enzyme action.
Explain how addition of base could inhibit enzyme action.

Experiment Lab 10 on Enzymes, Lab Reports of Biology

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Experiment 10 – Enzymes

Procedure

Questions