Lab 3: Biological Molecules & Enzymes
Objectives:
1. Understand the difference between an acid and a base
2. Know what the pH scale is and what it measures
3. Understand what is meant by a strong and weak acid
4. Know the four major categories of Biological Molecules. Understand what is meant by the term macromolecule.
5. Know what is indicated and what positive result looks like/ means in the following tests: Benedict’s Test, Iodine test, and Biuret’s test
6. Know the effects of pH and temperature on enzyme activity
7. Determine whether enzyme denaturation can be reversed
1. Acids have a pH lower than 7 and produce H+, bases have a pH higher than 7
2. a scale from 0 to 14 that measures how acidic or basic a solution is, with 7 being neutral. A pH less than 7 indicates an acid, while a pH greater than 7 indicates a base.
pH measures the concentration of hydrogen ions (H+) in a solution.
More H+ ions mean a higher acidity and a lower pH value.
Fewer H+ ions mean a higher alkalinity and a higher pH value.
3. A strong acid has a high concentration of H+ and a weak acid has a low concentration of H+
4. A macromolecule is a group of atoms bonded together
Carbs, proteins, lipids, nucleic acids
Carbs: provide quick energy. Ex: sugar, starch, glycogen, cellulose
Proteins: Build and repair tissues, enzymes, hormones, immune response. Ex: enzymes, antibodies, Hemoglobin, Muscle fibers
Lipids: Long-term energy storage, insulation, and cell membrane structure. Ex: fats and oils, steroids,
6. The colder it is it works slower, if it gets too hot it gets denatured and then it won’t work again. 7 pH is pure water, less than that has more H+ and is more acidic, more than 7 has more OH- and is basic.
7. Enzyme denaturation can’t be reversed.
BACKGROUND INFORMATION:
Biological Molecules: Nucleic Acids, Lipids, Carbohydrates and
Proteins
All living organisms consist of organic compounds (molecules containing the
carbon atom) and inorganic compounds. Water, an inorganic substance,
accounts for about 85% of the composition of a living cell. The bulk of the
remaining dry matter consists of carbon, oxygen, hydrogen and nitrogen.
These organic compounds are organized into four main types called
macromolecules: nucleic acids, carbohydrates, lipids, and proteins.
Carbohydrates and lipids contain the chemical elements carbon, hydrogen,
Polymer
Monomer
Monomer
Monomer
and oxygen. Proteins and nucleic acids contain these three elements plus
Monomer
nitrogen, sulfur and phosphorus (in the form of phosphate).
The large structures of many of the macromolecule groups are made of
many repeated simple molecules. In a sense, the assemblage of these
molecules resembles that of a necklace. The individual repeating molecules,
called monomers, are like the individual beads of the necklace. The string
used to hold the necklace beads together are similar to the bonds that link
together the individual monomers. Finally, the necklace as a whole (which is
comprised of many breads strung together) is analogous to a biological polymer, or a molecule made up of repeating monomers. Of the four major macromolecules, proteins, carbohydrates, and nucleic acids follow this general pattern. Although lipids have defining characteristics in their structure, they are not made of repeated monomers, differentiating them from the other three groups.
Polymers are built and destroyed by either joining together monomers (stringing together a longer necklace) or breaking apart monomers already bonded together (cutting the string on which your necklace beads are laced). The manufacturing of larger polymers from monomers is done via a process called dehydration. During dehydration, a water molecule is removed in order to facilitate the linking of monomers into larger polymers. Conversely, hydrolysis is a process that adds water molecules in an attempt to break larger polymers into smaller polymers.
Nucleic acids are made up of monomers called nucleotides. Each nucleotide consists of a five-carbon sugar (ribose or deoxyribose), a nitrogen base, and a phosphate group. There are two types of nucleic acids within cells: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These molecules are intimately involved in the production of
Nucleic acid structure Nucleotide
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proteins and the transfer of energy in and around the cell, and most importantly DNA serves as the means of transmitting genetic information to succeeding generations.
Lipids are a diverse group of chemicals that are classified together only because of their solubilities. All are insoluble in water but are soluble in the so-called fat solvents (ether, acetone, carbon tetrachloride). The simplest lipids (neutral fats) are composed of the subunits: glycerol (a three carbon sugar) and fatty acids. Fats and oils are extremely important in living systems because they serve as a concentrated storage source of energy (a fat molecule supplies more than three times as many calories as a carbohydrate or protein). One group includes the steroid hormones. Phospholipids all contain glycerol, two fatty acids and a phosphate group, and are the structural subunit used to form cell membranes.
Our studies in today’s lab will be focused on the remaining two categories of macromolecules: carbohydrate and proteins
Most of the substances that are called carbohydrates are classified either as monosaccharides, disaccharides or polysaccharides.
Glycerol
Fatty Acid Chain
Lipid Structure
Lipid Structure
Carbohydrate Structure Carbohydrate Structure
Monosaccharides are the simple (monomer) sugars (Glucose, fructose, and ribose would be examples of monosaccharides). Most of the monosaccharides found in living systems are molecules that contain either five or six carbon atoms. Disaccharides are sugars formed by the bonding together of two monosaccharides. For example, maltose is a disaccharide of two glucose molecules bonded together; and sucrose (table sugar) is a disaccharide of a glucose molecule bonded to a fructose molecule. Polysaccharides are polymers of many monosaccharides bonded together. For example, starch, glycogen, and cellulose are large molecules made up of many glucose molecules bonded together. Starch and glycogen are storage forms of carbohydrates; starch is usually formed in plants and glycogen in animals. They are insoluble in water and thus remain in cells until needed as a source of energy. Cellulose is an insoluble, structural polysaccharide that is a major component of the cell wall. In this exercise, you will perform two tests, Benedict's test and the iodine test, which can be used to identify carbohydrates.
From both structural and functional standpoints, proteins are extremely important macromolecules found within cells. Proteins are an important component of cellular membranes, serve as regulators of genetic material, form the contractile mechanism within cells, and serve as important structural elements. In addition, all of the enzymes (protein catalysts of metabolic reactions) in a cell are proteins. [It should be noted that some biological catalysts consist of RNA and are referred to as ribozymes.] Proteins are constructed of long chains of amino acids linked together by peptide bonds. The sequence of the amino acids in any given protein molecule is determined by the sequence of bases in the genetic material, the DNA, of the cell. By virtue of the large number of amino acids that comprise a single protein molecule, infinite numbers of combinations of different amino acids can be formed. The Biuret test (which determines whether peptide bonds are present) may be performed to identify proteins.
Although protein type and function is contingent on the specific
Monosaccharides Disaccharide
Polysaccharide
Protein Structure Protein
arrangement of amino acids, shape is also important. The amino acid strands that make up proteins are often folded into specific 3-D shapes that enable the protein to function properly. Any condition that causes a protein to lose its shape (we say that a protein who has lost its shape has been denatured) ultimately causes the protein to be non-functional. This can
Amino Acid
Amino Acid
Amino Acid
Amino Acid
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be very detrimental since proteins fulfill many roles in an organism. For
instance, many enzymes are used in the digestive process of animals. The readily digestible carbohydrates of the mammalian diet include starch (a
plant storage polysaccharide) as well as glycogen (an animal storage
polysaccharide). These polysaccharides are subject to hydrolytic cleavage (depolymerization) in the presence of amylases, enzymes that break down starch into smaller chains called dextrans and eventually all the way down to glucose monomers. In animals, saliva contains salivary amylase and the digestive juice from the pancreas contains pancreatic amylase. Denaturing the enzymes mentioned in the example above would inhibit the digestion of large carbohydrates (polymers) that are unusable by the cell to simple
sugars (monomers) such as glucose that can be readily used by the cell.
Factors that Affect Enzyme Function (Activity)
Enzymes will only function under certain environmental conditions. If
enzymes are subjected to slight deviations from optimal environmental
conditions, the enzyme may perform its intended function, but at a slower
rate. For instance stressed pancreatic amylase will break down less starch molecules than an amylase molecules under ideal conditions. Extreme
environmental conditions (large deviations from optimal conditions) can lead to the enzyme becoming denatured, which would render it completely
inactive. Temperature and pH are two environmental conditions that must be kept within a certain range, in order for enzymes to properly function.
The pH of a substance measures how acidic or basic a substance is. It is
based upon the concentration of hydrogen ions in a liter of acidic solution. The pH scale runs from 0 to 14. (The numbers below 7 refer to acid
solutions and the lower the number, the more concentrated the acid is.) The numbers above 7 refer to basic solutions and the higher the number the
more basic the solution is. The midpoint in the scale is 7, the pH of distilled water. At pH of 7, the concentration of hydrogen ions equals the
concentration of hydroxide ions. Any solution with a pH of less than 7 has
more hydrogen ions than hydroxide ions in solution. Conversely, any
solution with a pH of more than 7 contains fewer hydrogen ions than
hydroxide ions.
increasingly neutral increasingly
🡨🡨🡨🡨acidic basic🡪🡪🡪🡪
0 1 2 3 4 5 6 7 8 9 10 11 12 14 --------------------------------------------------------------------------------------------------------------------------------- 10-1 10-3 10-7 10-11 10-14
Concentration of H+ (in moles/liter)
🡨🡨🡨🡨Increasing H+ Concentration Decreasing H+ Concentration 🡪🡪🡪🡪
The pH scale is a logarithmic progression. This means that the numerical
values on the scale are not like an arithmetical progression, where the value of 2 is twice that of 1, or value of 3 is three times that of 1. Rather, numbers on the pH scale are based on powers to 10. On the pH scale, a pH of 2
indicates ten times fewer hydrogen ions than a pH of 1; and a pH of 3
indicates ten times fewer hydrogen ions than a pH of 2 and, a hundred times fewer hydrogen ions than a pH of 1.
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An acid is any substance that can donate a proton (hydrogen ion, H+). For example, in living systems carboxyl groups, COOH, are especially common proton donors. The carboxyl group dissociates in water to yield a proton and a negatively charged COO- ion.
CH3-CH2-COOH -----------------> CH3-CH2-COO- + H+ pyruvic acid pyruvate Hydrogen ion
A base is any substance that accepts protons (hydrogen ions, H+). Sodium hydroxide (a metallic hydroxide) is an example of a base. Upon ionization in water, sodium hydroxide releases a positively charged metallic ion and a negatively charged hydroxide ion:
NaOH ---------------> Na+ + OH-
(sodium hydroxide) (sodium ion) (hydroxide ion or hydroxyl ion)
Hydroxide ions are particularly effective bases because they have a very strong attraction for protons. Therefore, hydroxide ions readily pick up any proton, which may be in solution. However, it must be kept in mind that a base is any substance that accepts protons; and many compounds found in biological systems do not contain hydroxide groups but still accept protons.
The strength of an acid is determined by how readily it gives up protons, i.e., how readily the molecule ionizes. Some acids are strong acids, because they easily give off hydrogen ions (ex. hydrochloric acid, HCl). Weak acids do not give off hydrogen ions as easily (acetic acid, CH3COOH).
The strength of a base is determined by how readily it accepts protons. Some bases are strong bases (e.g., sodium hydroxide, NaOH), while others are weak bases (e.g., ammonia, NH3); again, mainly due to how readily the compound dissociates in solution and accepts protons.
Generally speaking, most enzymes require environmental conditions close to those exhibited in living organisms (i.e. 37° C and a pH of 7). Today, we will investigate the conditions of temperature and pH at which the enzyme amylase is most active.
Instruments/ ToolsVernier Monitor
Computer interface that is compatible with multiple attachments, that measure different scientific factors.
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In Class Exercises Exercise 1: Measurement of pH
Part A: Measuring Acids and Bases
Using both pH paper (aka. Litmus Paper) and a pH meter, determine the pH of each of the following:
a. 0.1 M HCl
b. 0.1 M NaOH
c. Unknown 1
d. Unknown 2
e. Unknown 3
1. First fill the corresponding beaker with approximately 20 mL of solution, e.g., pour 0.1 M HCl into beaker labeled 0.1 M HCL
To test a solution with pH paper just dip one end of the paper into the
solution sample, and then compare the color of the paper with the color chart on the container which holds the pH paper. Use the expensive pH paper sparingly. The pH-meter needs to be kept in buffer solution when not in use and washed off with distilled water before it is placed in a solution. Dip the pH probe into the beaker of solution you want to test. Wait for the result to stabilize then record the number in the table to the right. Use the provided distilled water wash bottle to rinse the pH probe over the beaker labeled “rinse pH probe” before dipping the probe into the next solution. After all your results have been recorded, rinse the pH meter with distilled water and screw on the pH buffer solution cap. The results for the three unknown substances will be used later on in the lab, so be sure to keep these results in a safe place.
Part B: Strong Acids
Using both pH paper and a pH meter, you will determine the pH of 0.1 M (acetic acid). This solution has the same concentration as the 0.1 M of hydrochloric acid (HCl) that you measured in part A of exercise one.
1. Measure the pH of 0.1 M CH3OOH using both the meter and pH
paper. Notice that the pH is not the same as the HCl you measured
previously. Why do you think the pH of these two substances are
different?
Would a greater concentration of HCl have a lower pH? Why?
____Yes, a greater concentration of HCl will have a lower pH because it increases the concentration of hydronium ions (H₃O⁺) in the solution___
What is the magnitude of difference in regard to the number of hydrogen ions Unknown 1 and Unknown 2?
_____________________________________________________
Instruments/ Tools Litmus Paper
Vernier pH Attachment Determines pH of solution.
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Which of the unknown substances has the greatest concentration of hydroxide ions?
____________________________________________________
Which of the unknown substances has the greatest concentration of hydrogen ions?
____________________________________________________ Exercise 2: Determining the Presence of Proteins
Proteins are polymers of amino acids that are linked together by peptide bonds. The Biuret test contains a chemical reagent that binds specifically to peptide bonds; thus, the Biuret reagents test for the presence of proteins. Proteins are indicated by a violet color.
1. Obtain a vial of amylase (high concentration), an enzyme used to break down starch into glucose, from your instructor. Pipette 3 mL of the amylase (using the correctly labeled pipette at your table) into a clean test tube.
2. Then using the pipette labeled 1 M NaOH, add 3 mL of NaOH to the test tube.
3. Finally, add ~15 drops of 0.5% copper sulfate (CuSO4) to the test tube and gently shake tube (being sure not to spill the liquid).
What color is the solution? _____________________________________ What does this tell you about amylase?
___________________________________________________________
Exercise 3: Effect of pH on Enzyme Activity
As mentioned, enzyme activity is influenced by environmental conditions such as pH and temperature. In this experiment, we will be determining the optimal pH for amylase activity (i.e. determining the pH in which amylase works best). We will be assessing amylase activity based off its ability to break down the polymer starch into its monomers, glucose. Since we want to know if amylase was able to break down starch, we could measure either the amount of starch present (using an iodine test) or the amount of glucose present (using a Benedict’s test).
An iodine test can be used to determine the presence of starch. Iodine, which is naturally a light brown color, changes color when starch is present. Since starch is broken down when amylase is functional, the presence of starch indicates amylase is not functional and the absence of starch indicates amylase is functional.
Instruments/ Tools Pipette
Instrument that measures volume of a liquid; most commonly used when
measuring in mL.
Starch [Polymer] Functional Amylase
Glucose [Monomers]
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Benedict’s test can be used to detect the presence of reducing sugars (e.g. glucose and many other mono- and di- saccharides). The blue Benedict's solution contains copper ions which combine with most simple sugars, such as glucose, to form a precipitate (solid). Depending on the concentration of the sugar, various colors develop from green to yellow to orange to red. A yellowish green indicates a low concentration whereas an orange-reddish color indicates a high concentration of sugar. Therefore, the Benedict's test can provide both qualitative and quantitative information. The Benedict’s test requires a heat catalyst. An orange to red opaque color indicates a strongly positive Benedict’s test result, indicating a high concentration of reducing sugar is present. If a reducing sugar is not present, a blue, clear color results (essentially the color of the Benedict’s reagent alone) indicating a “negative Benedict’s test result.”
A. Determining Positive and Negative Controls
In lab 1, we discussed the importance of having a control group to act as a comparison for the treatment groups. In most experiments, the control groups are negative controls, showing the expected results if a treatment is not added, or a reaction does not occur. Although negative controls are critical in most controlled experiments, positive controls are also often used. A positive control shows what results should be expected when a particular treatment or reagent is added, and is functioning properly. In a sense, a negative control illustrates the outcome you should expect when something is not occurring, and a positive control shows the result you should expect if something is occurring optimally. To help us interpret the results of the iodine test and benedict’s test, we are going to generate a positive and negative control for each test.
Before determining how to generate a positive and negative control, we first need to consider what outcomes we should expect if amylase does or does not function.
If you add starch into a test tube with amylase, and the amylase is functional, what should be left in the tube at the end?
_______________________________________________________
If you add starch into a test tube with amylase, and the amylase is NOT functional, what should be present in the tube at the end?
_______________________________________________________
Given what you just determined, what should you use as a positive control?
Given what you just determined, what should you use as a negative control?
Instruments/ ToolsGlucose [Monomers] Transfer Pipette
Instrument that allows small quantities of liquid to be easily transferred from one container to another, without spilling. Does not allow user to precisely quantity volume of liquid transferred.
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Setting up Iodine Test Controls:
1. Using a transfer pipette, place a couple of drops of the substances you determined above, into two
wells of the porcelain dish at your desk. Be sure to
label which is your positive and which is your
negative control, using the wax pencil at your desk.
2. Add a drop of iodine into each well, and record the colors in the space below:
+ Control Color: _____________
--Control Color: ____________
Setting up Benedict Test Controls:
1. Using the correctly labeled pipette tip, pipette 5 mL of the solutions you outlined above, into two different test tubes. Be sure to label which is our positive and which is your negative control, using the wax pencil at your desk.
2. Add 8 drops of benedicts solution into each test tube.
3. Place the both test tubes into a hot water bath (around 100°C) for roughly 8 minutes.
4. Remove the test tubes with a test tube holder (caution, the glass will be hot) and record the colors in the space below:
+ Control Color: _____________
--Control Color: ____________
B. Determining the optimal pH for Amylase
1. At your desk you will have 3 shorter clean test tubes. Label the 3 test tubes as follows: 1,2, and 3 (the numbers correspond to the three unknown pH solutions tested in exercise 1).
2. Pipette the following into each of the test tubes:
Tube 1: 2 mL of amylase; 5 mL of unknown 1
Tube 2: 2 mL of amylase; 5 mL of unknown 2
Tube 3: 2 mL of amylase; 5 mL of unknown 3
3. Gently swirl all test tubes ensuring the solution and amylase are well mixed. Allow all test tubes to sit for 5 minutes.
4. After the test tubes have sat for 5 minutes, add 2 mL of 2% starch solution to each test tube and wait another 5 minutes, gently stirring the liquid.
5. After 5 minutes, use a transfer pipette to add a few drops of liquid from each test tube, into the porcelain dish that contains your positive and negative iodine test controls. Be sure to remember which liquid is in each well, by using the wax pencil at your desk to label the wells.
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6. Add 1 drop of iodine (I2Kl) to each of the three wells, and record
the color for each of the corresponding pHs. Indicate whether the
color indicates if starch is present, and if the amylase is
functional.
** Your instructor has prepared the same tubes outlined in step 1 using Benedict’s test. Record the Benedict Test results in the table below as the instructor shares them.
Test Unknown 3
Iodine Test
Benedict’s Test
Outcome
Unknown 1
Unknown 2
Color
+ or – for starch?
Amylase Functional?
Color
+ or – for glucose?
Amylase Functional?
Based on your observations from the Benedict’s test and iodine test, what is the optimal pH for amylase function? How can you tell
_________________________________________________________________________________________ _________________________________________________________________________________________ _________________________________________________________________________________________
Exercise 4: Effect of Temperature on Enzyme Activity
Temperature can influence enzyme activity. In this exercise, your group
will determine the optimal temperature for amylase, from the following
treatment groups:
0-4°C (Ice bath)
37°C (Water bath)
95°C (Hot Plate Water Bath)
1. Remove 3 clean tall test tubes from your test tube rack
and label them S (this stands for starch) 0, S37, & S 95.
2. Remove 3 short test tubes, and label them E (this stands
for enzyme) 0, E 37, and E 95.
3. You will add your solutions from this exercise, to the
porcelain dish you used in exercise 3. Using the wax
pencil on your bench label the wells on your dish, to match
the image below.
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4. Shake the bottle containing 2% starch solution, using your “starch” pipette, place 4 mL of this solution into all the test tubes labeled S.
5. In your test tubes labeled E, place 2 mL of fresh amylase using the pipette labeled amylase.
6. Place the two tubes (S & E) 0 in the ice chest on the side counter, the tubes labeled 37 in the beige water bath, and the tubes labeled 95 in the pot on the hot plate.
7. Allow all tubes to sit for 15 minutes.
8. After the 15 minute incubations, carefully pour (the contents of the amylase tube (labeled E) into the corresponding starch tube (labeled S). Quickly, but carefully, remove some of the mixture from each test tube and add it to the appropriate wells in your porcelain dish, using a transfer pipette.
9. Add a drop of iodine to each well and record your results in the table below.
Caution!
The 95°C water bath will burn your skin. Use test tube holders, or pot holders to remove your test tubes from this treatment after the 15- minute incubation period. You can use your bare hands to remove the tubes from the other treatments.
Color
Outcome 95°C
0°C
37°C
+ or – for starch?
Amylase Functional?
10. Five minutes after you run the iodine tests outlined in step 9, rerun the solutions placed in the 0°C and the 95°C water bath, by placing some of the unused solution in the test tube, in an unused well, and adding a drop of iodine solution.
How does the new 0°C iodine test look, compared to your initial iodine test? What does this indicate about amylase’s function in this condition?
__________________________________________________
How does the new 95°C iodine test look, compared to your initial iodine test? What does this indicate about amylase’s function in this condition?
__________________________________________________
Based on the data collected above, which temperature is ideal for optimal amylase function? How can you tell?
__________________________________________________
Why was the starch and enzyme initially split into two different test tubes? Why not just combine them, prior to placing them in the water bath?
__________________________________________________
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Lab 4: Molecular Motion
__________________________________________________