LAB #3 - ORGANIC COMPOUNDS OF BIOLOGICAL SIGNIFICANCE

 

 

Section I A   Molecular Hierarchy - Organic and Inorganic Chemicals

 

[2]  Hi, this is Pat Farris again.  I see you survived last week's chemistry lab and are ready for more.

Organic chemistry is a huge field of study.  Your body is composed of organic compounds, you eat organic compounds and you are surrounded by organic compounds.

 

[3]  As a distinct field of study, organic chemistry had its beginning during the mid-1800's.  It was during this time that a German chemist, Friedrich Wohler, synthesized the first organic compound using inorganic materials.  The compound he synthesized was urea--a constituent of urine.  While I realize that this may not sound terribly exciting to you, its synthesis made a tremendous impact on the scientific community.  Until this time, it was believed that organic compounds could not be manufactured synthetically from inorganic materials.  Wohler demonstrated that organic compounds could indeed be made in the laboratory.

 

[4]  Today, organic chemistry is a field of immense importance to technology:  it is the chemistry of medicine and biology, plastics and drugs, superconductors and petroleum, pesticides and nutrition.  Aside from water, living organisms are made up chiefly of organic compounds and all the biological processes we will talk about this semester are ultimately a matter of organic chemistry.

 

[5]  Let's first clear up some misconceptions about the term "organic".  Some people think the word organic means wholesome, natural or healthful.  I can assure you that thousands of organic substances are neither natural nor healthful.  Plastics and insecticides, for example, are organic compounds, but you probably would not consider them to be wholesome or natural.  What, then, do we mean when we say a substance is organic?

 

[6]  With few exceptions, organic compounds are molecules composed of carbon and hydrogen.  Other elements like oxygen, nitrogen, sulfur and phosphorus are often present as well.  So, compounds containing carbon and hydrogen are called organic compounds.

 


[7]  Compounds that do not contain carbon and hydrogen together are called "inorganic".  Water, oxygen, sodium chloride and sodium nitrate are some common inorganic substances.  Turn to page 3.3 in your lab manual and summarize the difference between organic and inorganic chemicals and complete Table 1 before you click on the continue button.

 

[8]  Not only are there chemical differences between organic and inorganic compounds, they are used differently by living organisms.  Inorganic compounds are used to construct simple organic building blocks.  Fairly simple inorganic compounds such as carbon dioxide, water and inorganic nitrogen compounds are combined to form organic building blocks known as amino acids, simple sugars, fatty acids and nucleotides.

 

[9]  These small organic building blocks, in turn, are used to construct proteins, polysaccharides, lipids and nucleic acids, respectively.

 

[10]  Take a moment to examine the relationship between the building blocks and the larger compounds.

 

[11]  A few inorganic substances are used in minute quantities but play essential roles in the formation of certain organic compounds.  Two such substances are iron, which is used to complete the structure of hemoglobin; and magnesium, which is required for chlorophyll.  Although the quantity of magnesium in a plant or iron in a human is relatively small, these elements play a critical role in the organism's well-being.

 

[12]  Organic compounds constitute the vast majority of an organism's dry weight, that is, if you were to remove all the water from an organism.  All of the cell's structural material is organic.  Every chemical reaction occurring in the cell is controlled by organic molecules called enzymes.  So, organic compounds not only serve as major structural material, but control all the reactions in your body as well.

 

 

Section I B    Synthesis of Complex Macromolecules

 

[13]  Now that you understand some of the chemical and functional differences between inorganic and organic chemicals, let's examine how your body makes and utilizes organic molecules.

 

[14]  We can discuss chemical reactions in terms of energy relationships.  Reactions can be "endergonic" if they store energy, or "exergonic" if they release energy.

 

[15]  Chemical reactions can also be classified on the basis of molecular complexity.  If the products of the chemical reaction are bigger and more complex than the reactants, the reaction is classified as anabolic.  If, on the other hand, the products are smaller and less complex than the reactants, the reaction is classified as catabolic. 

 


[16]  Note the first relationship between amino acids and proteins.  Hundreds of relatively small organic molecules called amino acids can be used to make one large complex molecule called a protein.

 

[17]  Just as bricks can be used to construct walls and buildings of many different patterns, amino acids can be used to construct many different kinds of proteins.

 

[18]  Simple sugars called monosaccharides, can be combined to make complex carbohydrates called polysaccharides.  The third example shows that fatty acids are required to make complex lipids and finally, many nucleotides are required to make a nucleic acid like DNA.

 

[19]  These kinds of chemical reactions are called anabolic.  Anabolic chemical reactions always result in products that are bigger and more complex than the reactants.  So a cell can start with some amino acids and connect them together to form a complex protein molecule composed of hundreds of amino acids.

 

[20]  Living organisms, of course, can also break down large, complex molecules.  The reactions that break down large molecules into their smaller building blocks are called catabolic reactions.  Catabolic reactions serve two purposes.  First, they release energy for metabolic work and second, they serve as a source of raw materials for anabolic processes. 

 

[21]  During the digestion of a steak, for example, you break down proteins into amino acids and fats into fatty acids.  Then, through anabolic reactions, you reassemble the same amino acids into your own kind of protein molecules and fatty acids into your own kind of fat molecules.  The difference between cow protein and human protein is not in the kinds of amino acids present but the way the amino acids are arranged.  We'll get back to that idea later on in this lab.

 

[22]  Another way to describe these organic reactions is in terms of water.  These two molecules are reacting to form a more complex molecule and one water molecule.  Examine the two reactants and you will see where the water comes from.  Note that the "OH" comes from one reactant and the other "H" comes from the other reactant.  When a water molecule is formed during an anabolic reaction, it is called a dehydration since water is being removed.

 

[23]  Here's another anabolic reaction that involves dehydration.  Do you see where the water is coming from?

 


[24]  Anabolic reactions involve the removal of water, catabolic reactions involve the addition of a water molecule.  This is a typical catabolic reaction.  Here a large complex molecule is broken down into two smaller building blocks.  Notice that a water molecule is one of the reactants.  Upon closer examination of the products, you should be able to see where this water molecule went.  Because water is required to break apart this molecule, the reaction is called "hydrolysis".  The prefix "hydro-" means water and the suffix "-lysis" means to break apart.  So the term hydro-lysis, (hydrolysis) means to break apart with water.

 

[25]  All of these catabolic reactions involve the addition of water.  Later on, you will have an opportunity to actually perform hydrolysis and dehydration using molecular models.  Right now, I'd like you to complete part B on page 3.3.  After completing question number 2, click on the continue button.

 

[26]  Let's check to see if you understand these organic reactions.  Click on the correct answer.

 

IF CORRECT RESPONSE: [27]  Good work!  This is an example of a catabolic reaction since the products are smaller, and hydrolysis since water was used during the reaction. Let's go on!

 

 

Section II  Carbohydrates

 

[28]  Now that you understand the chemical reactions related to organic compounds, let's look at each category in a little more detail.  There are four major groups of organic compounds, and we'll have time this week to look at three of them.  DNA, a nucleic acid, is so important that we'll save it for a later lab.

 

[29]  Let's look at the relationship between the number of carbon, hydrogen and oxygen atoms in a simple carbohydrate.  It was originally believed that carbohydrates were simply carbon skeletons to which water molecules were added.  Because of this early belief, these substances were named "carbohydrates" or "hydrates" of "carbon".

 

[30]  Although many carbohydrates contain other elements besides carbon, hydrogen and oxygen, we can always look for this general formula and ratio to characterize carbohydrates.  Remember that there are exceptions.  Here you can see that the three common elements in carbohydrates appear in a 1:2:1 ratio.  Compare this ratio with the four examples given to you on page 3.4.  This ratio of elements is one that you will want to remember, since for our purposes, it will help you recognize the carbohydrates.  When you have filled in the answers on page 3.4, click on the continue button.

 

[31]  Now you get to build one of my favorite molecules, glucose.  It's easiest to start with the basic ring of carbon, then add the hydrogens and oxygens from there.  Take your time and look for the CH2O arrangement that you just learned.  Click on the continue button when you have completed your molecule.

 


[32]  We'll get back to your fabulous glucose molecule in a little while, but for now, turn to page 3.5 and study the table listing the five common carbohydrates and the role each plays in the cell.  Then, click on the continue button.

 

[33]  Did you notice that some carbohydrates are made up of hundreds of smaller carbohydrates?   Let's define the levels of carbohydrate complexity.  In Part C-1 of your lab manual you will see the terms monosaccharide, disaccharide and polysaccharide.  Fill in the definitions as we go.  Monosaccharides are simple sugars-- like deoxyribose, glucose and fructose.  For the most part, these simple sugars are used as building blocks or as a fairly quick source of energy.  They're easy to recognize by their single ringed structure.

 

[34]  Disaccharides are a little bigger and more complex because they are composed of two covalently bonded monosaccharides and have a two ringed structure.  Sucrose is a disaccharide composed of glucose and fructose.  Lactose, or milk sugar, is another disaccharide but it is composed of one glucose molecule and one galactose molecule.  Although plants use disaccharides for transporting carbohydrates around, humans find little use for this type of molecule at the cell level.

 

[35]  Polysaccharides are huge carbohydrate molecules composed of many hundreds of covalently bonded monosaccharides.  Polysaccharides like starch and glycogen are used for long-term energy storage.  Other polysaccharides like cellulose and chitin are used for structural support.

 

[36]  Now I will have you construct a disaccharide incorporating the glucose molecule that you constructed previously.  Complete the carbohydrate section on pages 3.5, 3.6 and 3.7.  Have your molecule checked by an instructor and be prepared to tell him or her how you constructed it from the two monosaccharides.

 

[37]  When you have finished your test for carbohydrates and drawn some leucoplasts from the potato sample, click on the continue button.

 

 

Section III A. - C.    Lipids

 

[38]  The lipids constitute the second major group of organic compounds.  While the term "lipid" may be unfamiliar, many common everyday substances belong to this group.  Fats, oils and waxes are some familiar lipids.

 

[39]  On page 3.7 record the chemical definition of a lipid.  Also, take a look at the table of common lipids in your lab book and the roles they play in the living organism.

 

[40]  Now it's time to see how lipids are constructed.  Here you can see an abbreviated version of a triglyceride, showing the shapes of the smaller pieces.  Let's look at its structure in some detail.

 


[41]  Here is a completed triglyceride structure that you can copy at the top of page 3.8. 

[42]  Let's look at each component of a triglyceride in detail.  The "backbone" of the molecule is called glycerol, and it serves as the attachment point of three fatty acids.  There are several structures of fatty acids, each having unique properties.

 

[43]  Examine this fatty acid.  If you will consider the ratio of carbon, hydrogen and oxygen atoms present, you will notice the small amount of oxygen present.  Relative to carbohydrates, the number of oxygen atoms in a fatty acid is considerably lower.  This is one of the important characteristics that differentiates lipids from carbohydrates and will help explain why lipids act as they do.

 

[44]  When hydrogen covalently bonds with carbon, a very strong bond is formed-- a bond considerably stronger than a carbon to oxygen bond or a carbon to carbon bond.  Because fatty acids contain such a large number of hydrogen to carbon bonds, these molecules possess a considerable amount of energy.

 

[45]  Here you see another fatty acid.  As you look down the long chain, notice that each carbon atom in the chain has two hydrogen atoms attached.  Such fatty acids are said to be "saturated" because they contain the maximum number of hydrogen atoms.

 

[46]  This fatty acid molecule contains one double covalent bond between adjacent carbon atoms.  As a result of the double covalent bond, this fatty acid is NOT saturated with hydrogen atoms and is referred to as an "unsaturated fatty acid".  Sound familiar?  If you're interested in nutrition, it's a term you've probably used before.

 

[47]  This fatty acid contains three double covalent bonds between adjacent carbons.  Because this fatty acid contains more than one double covalent bond, it is called a "polyunsaturated fatty acid".

 

[48]  The degree of hydrogen saturation will affect the physical properties of the triglyceride.  Those that are composed of saturated fatty acids are solid at room temperature and are referred to as fats.

 

[49]  Those triglycerides composed of unsaturated fatty acids are liquid at room temperature and are referred to as oils.

 

[50]  Let's try a question about fats and oils.

 

[51]  Let's try another.

 

[52]  Steroids represent another general type of lipid.  Here you see the general structure of a steroid.  Copy this general shape in the space provided on page 3.8.


[53]  Although steroids appear radically different from triglycerides, like all lipids, they are insoluble in water but soluble in organic solvents.  Note that all of these steroids possess the general four rings of carbon.  You don't need to copy these down but you should be able to recognize the general configuration.  One steroid differs from another because of the kinds of atoms or groups of atoms attached to the basic carbon skeleton.  These differences can be great-- for example, between cholesterol and testosterone, or slight-- as between testosterone and estrogen, the male and female sex hormones respectively.

 

Section III  D. Transporting Lipids

 

[54]  Triglycerides also have an important role in cell structure.  With a slight but significant modification, the last fatty acid is replaced with a phosphate group and a phospholipid is formed.  Phospholipids are a major component of all plasma membranes- the thin layer of molecules surrounding the cell and separating it from its environment.

 

[55]  As you know, living organisms are mostly water.  But how do organisms move lipids around the body if they are only soluble in nasty things like chloroform and benzene?  This can be done by enveloping the lipids in chemicals called emulsifying agents.

 

[56]  Let's look at these emulsifying agents in a little more detail.  All emulsifying agents are molecules that have two distinct ends - one end is hydrophobic or "water fearing" while the other is hydrophilic or "water loving".  The hydrophobic end points away from water molecules while the hydrophilic end points toward water.  Copy this structure in your lab book.

 

[57]  If you had a jar with both oil and water in it, you'd notice that the two substances would always separate.  Oil is a hydrophobic substance.  Even if you shook up the solution, they would separate again.

 

[58]  But if you add a little bit of an emulsifying agent and shake it up a bit, a remarkable reorganization takes place.  Each oil droplet that was generated by the shaking is now completely surrounded by several molecules of emulsifying agent.  The hydrophobic ends stick into the oil droplets and the hydrophilic ends stick into the water.  They will stay this way for a very long time, forming what we call a stable emulsion.  By forming small organized structures, lipids can be dispersed as little droplets in water, something they would ordinarily NOT do.  For living organisms this stable dispersion allows lipids to be transported around the cell and throughout the body.

 

[59]  One common, naturally occurring emulsion is milk.  I'd like you to examine the fat globules in cow's milk under the microscope.  This will be a real test of your microscope skills, by the way.  Tiny white droplets are very hard to focus on.  You will have to adjust the iris diaphragm very carefully.  When you have completed all of page 3.8, we'll go on to proteins.


Section IV A. - C.   Proteins

 

[60]  You did clean off your milk slide and cover slip, didn't you?  Great!  Let's go on.  The third group of organic compounds, the proteins, are involved with virtually all of the events that occur in living organisms.  On page 3.9, record this definition and take a look at the examples of proteins given in the table.

 

[61]  Proteins are constructed from just 20 basic building blocks called amino acids.  The amino acid molecule itself is rather simple.  In the space provided on page 3.9, copy the general structure of an amino acid.  All 20 amino acids have this fundamental structure.  Each has an amino end and an acid or carboxyl end.  The letter "R" is used to designate a variable side chain.  This means that this is where each of the 20 amino acids demonstrates its uniqueness.

 

[62]  Here you see two amino acids-- tryptophan and glutamic acid.  Although both are chemically different, note that each has the basic amino acid components-- the amino and carboxyl ends.  Their uniqueness comes from their "R" groups.

 

[63]  Here you see some more amino acids.  Maybe I should have you memorize these...  Oh well, maybe next semester.  For now, just notice that each has the same basic structure that you drew on page 3.9, but show variability in their "R" groups.

 

[64]  You should, however, be able to distinguish an amino acid from a triglyceride, steroid or carbohydrate-- even if the amino acid is one that you've not seen before.  Just look for the common components, the amino and carboxyl ends.

 

 

 

Section IV  C. 3. and D.   Levels of Protein Complexity

 

[65]   Proteins are so complex, we need to discuss several levels of complexity as we go.   They are called the primary, secondary, tertiary and quaternary levels of protein complexity.  Take a look at Table 6 on page 3.10.

 

[66]  The primary structure of a protein is its amino acid sequence.  Although each of the thousands of proteins you manufacture in your cells use the same 20 amino acids as their original building blocks, each protein is unique because of the sequence of amino acids in the molecule. 

 


[67]  If we, or our cells, start to build a protein such as insulin, we need to know the exact sequence of amino acids.  You might be wondering how a cell "knows" in what order to put the amino acids.  The answer is DNA.  Your genes contain the "recipe" for making thousands of proteins.  As you will see, it's the sequence of amino acids that determine the final function of the protein.

 

[68]  When our chain of amino acids is long enough, it begins to take on a characteristic shape.  This characteristic shape is called its secondary structure.

 

[69]  Here you see two common secondary structures:  the alpha helix and the beta pleated sheet.  The alpha helix is shaped like a coil and it is characteristic of proteins that make up hair, fingernails and wool.  The beta conformation resembles a folded piece of paper, and is characteristic of silk fibers and spider webs.

 

[70]  The secondary structure of a protein results from the interactions between adjacent amino acids.  The conformation of the secondary structure will depend on the order of the various amino acids in the chain.

 

[71]  As the amino acid chain grows longer, the coiled chain often begins to fold back on itself.  This folding and bending of the amino acid chain is quite independent of its secondary structure and is known as its tertiary structure.  The tertiary structure of a protein is the most important, since it is the tertiary structure that determines what the protein can do. 

 

[72]  Remember how important the lipids were to the structure of a membrane?  Well, the other important component in a plasma membrane is the assortment of proteins.  Each protein has a distinctive structure, giving each a distinctive role in the life of a cell.  Notice that proteins can have hydrophobic and hydrophilic regions, just like the phospholipids did.

 

[73]  Here you see the tertiary structure of a hypothetical enzyme.  The chamber where the two reactant molecules fit in is called the active site.  The shape of the active site results from the tertiary structure of the enzyme.  Once the reactants fit into the active site, the enzyme forces them to react.  The products are then released, freeing the active site for more reactants.  Now you can see that if the enzyme's active site were changed in any way, the enzyme would no longer be able to catalyze the reaction because the reactants would no longer fit into the active site. 

 

[74]  Some proteins are sooo complex that they even have what is called a quaternary structure.  This is when a protein is composed of two or more amino acid chains.  Our insulin molecule has this level of complexity, since it has two chains of amino acids that fit together.

 

[75]  Here you see a very elegant chemical called hemoglobin.  One molecule of hemoglobin is composed of four amino acid chains, so it's also a nice example of a tertiary structure.

 

[76]  To sum up these levels of complexity, I'd like you to examine the display that has been set up in front of the lab.  In your own words, summarize these different levels of protein complexity.  When you have completed the Table, click on the continue button.


[77]  How'd it go?  I hope you appreciated the three dimensional nature of proteins and used all the right terminology for your descriptions, such as amino acid sequence, alpha helix and folding.  Now you can finish Part D completing the section on how amino acids combine to form proteins.

 

Section IV  E.  Effect of Temperature

 

[78]  This week's experiment will have you examine the effect of temperature on the secondary and tertiary structures of an enzyme.  The reaction we will use is the same reaction you studied last week--the pyrocatechol reaction using tyrosinase as the enzyme.  Just to refresh your memory, recall that pyrocatechol, one of the reactants, is colorless.  When pyrocatechol reacts with oxygen inside the active site of tyrosinase, a yellow product called quinone is formed.

 

[79]  One thing I'd like you to determine today is the degree of enzyme activity.  This can be determined indirectly by estimating the relative amounts of quinone produced.  The yellower the final solution, the greater amount of quinone present.  The greater the amount of quinone, the higher the enzyme activity.

 

[80]  Before you actually start the experiment, let's review protein structure, since tyrosinase is a protein.  Remember from Table 6 that it was the tertiary level of complexity that determines the shape of the active site.  If anything changes the active site, the enzyme will not work.  This permanent change in the structure is called denaturation.  Make a note of this term, as it is the basis for your experiment.

 

[81]  Denaturation is not just some weird laboratory process, it happens every day.  As you know, eggs contain protein.  Right out of the shell, egg white is a gooey, clear fluid.  After heating, however, the protein undergoes denaturation resulting in a physical change.  The egg white becomes a solid, white mass.  This is because the tertiary and secondary structures have changed.

 

[82]  Now we're ready to test tyrosinase activity.  A couple of hints before you get started.  When tyrosinase is heated, it will often form a pink color.  This color should be ignored since it is not an indication of quinone formation.  Remember, if it ain't yellow, it ain't quinone.  The yellow color can be quite subtle at first, so remember to look down through the tube to look for quinone.

 

[83]  When you have completed the entire experiment and have had your graph signed and answered all the questions on page 3.12,  click on the continue button and I'll have some questions for you.

 


[84]  Let's review what you've learned about enzymes during the last two weeks.  Enzymes are proteins with a very complex structure.  Their structure determines their function, since it determines the shape of the active site.  Tyrosinase can only function, that is, form quinone, under certain conditions.  Do you remember what these conditions were?  Last week we looked at the effect of pH and this week we tested temperature.  What happens under unfavorable conditions?  The active site is permanently altered.  Now let's try some questions. 

 

[85]  Click on the correct answer.

 

[86]  That's correct!  Thanks for paying attention!  Let's try one more.

 

[87]  That's terrific, you're turning into quite the biochemist.

 

[88]  Now if you would take a minute to make sure the booth, microscope slides and all of your test tubes have been cleaned up, you'll be free to go.  Click on the main menu button and we'll see you next week.