The Chemical Aspects of Life

BIOLOGY 1001 FALL 2004

LABORATORY 1: 9/13 – 9/17 The Chemical Aspects of Life

Organisms and cells operate as chemical factories. In order to understand life, we must understand biochemistry. We need to learn what chemical compounds are found in living organisms, appreciate how they are studied, and understand how they operate in cells.

One striking fact is that living organisms consist primarily of water! On the average, 85% of the weight of a cell is water. The remaining 15%, the dry matter, must exert control over all that water. The bulk of the dry matter consists of organic compounds--substances built of carbon chains. The most common organic compounds are of four classes: proteins, carbohydrates, lipids (fats and oils), and nucleic acids. Only 1% of the total weight of a living organism is composed of inorganic materials such as potassium, calcium, iron and magnesium.

Organic compounds are generally found as monomers (groups of one), dimers (groups of two), and polymers (groups of “many”). The same four classes of organic compounds are found in all living organisms. Although the monomers are generally the same in all organisms, different kinds of organisms make and use different kinds of polymers for different functions.

Table 1. Four classes of organic compounds and their monomers, dimers, and polymers. monomer dimer polymer(s) amino acid dipeptide (examples: serine, protein = polypeptide (example: aspartame/Nutrasweet) proline, and glycine) starch (energy storage in plants) monosaccharide disaccharide (examples: sucrose, glycogen (energy storage in animals) (examples: glucose lactose, maltose, and cellulose (structural support in plant and fructose) sucrlose/Splenda) cell walls)

fatty acid uncommon fats, lipids (example: oleic acid)

nucleotide DNA Uncommon (example: ATP) RNA

Proteins, carbohydrates and lipids are colorless and can be difficult to study. One way to detect them is to produce a colored product in a chemical reaction. For example, the Biuret reaction with proteins produces a certain color. The appearance of this color can qualitatively tell us protein is present in a sample. A determination of the intensity of the color may give us some idea of the amount of protein. In order to conduct a quantitative test to give a specific measurement of protein, we would need to use a spectrophotometer to quantify amounts of color (we won’t be doing this today).

Lab 1 - 1 Color reactions can also tell something of the nature of the molecules present. For example, amino acids linked into proteins (polypeptides) produce a violet color with the Biuret reaction; free amino acids produce a pink color. The Benedict's solution test will produce a color with monosaccharides but not disaccharides.

In this laboratory, we will utilize chemical tests to identify the presence of proteins, carbohydrates and lipids in qualitative assays. You may wonder why we are ignoring nucleotides, DNA, and RNA today. While there are tests to identify these chemical compounds, they are very specific. For example, we could do a test to determine whether there is ONE SPECIFIC gene (a specific sequence of nucleotides) in a sample. If you take BIOL BC1002 next semester, you will use this test, PCR (polymerase chain reaction), to identify specific genes found in different bacterial viruses.

Complex organic molecules A. Proteins B. Carbohydrates C. Lipids

TESTING AN UNKNOWN

For each test you perform today, you will also be testing an unknown solution. Record your results in the appropriate tables as you work through the lab today.

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Textbook reading prior to coming to lab: Tobin and Dusheck: pp. 40-63.

Before starting this exercise, refer to the section on lab safety on page v of the syllabus.

You will be working in pairs today.

Conducting an experiment: Whenever a scientist conducts an experiment, she or he must design the experiment carefully. Suppose she is testing for the presence of the causative agent of Mad Cow Disease in different samples of ground beef. Hypothetically, she mixes her Test Reagent with samples 1, 2, and 3. Her results are a green color for these three samples. Can she conclude that they are all contaminated? Of course not, she needs to have CONTROLS in her experiment. Ideally, she should have a positive control (one that is known to give a positive result) and a negative control (one that is known to give a negative result). When she tests her controls, she finds the negative control gives a green color and the positive control gives a red color. What conclusions can she make about the results of her samples of ground beef? Every time YOU do an experiment, you should use both positive and negative controls.

Lab 1 - 2 COMPLEX ORGANIC MOLECULES

A. PROTEINS

Proteins are made up of long chains of amino acids. Each amino acid is made up of a central (alpha) carbon atom, to which four different chemical groups are attached. The first three of these + - groups are the same for all amino acids: H3N , COO , and H. The fourth chemical group is variable and is called the “R” group, or side chain. Some R groups are basic, some are acidic, some are polar and uncharged, and some are nonpolar (see Table 3-5 on page 58 of your textbook). The identity of the side chains, and the order in which they are joined together to make a protein, makes the structure and function of each protein unique. Some familiar proteins include antibodies, hormones, enzymes, collagen (found in skin, muscle, bones, tendons, and ligaments), and keratin (found in hair and nails).

Amino acids are joined together by peptide bonds to form polypeptides (a synonym for protein). The Biuret reagent reacts with compounds containing two or more peptide bonds. The Biuret reagent contains copper ions that complex with peptide bonds in an alkaline solution to form a purple precipitate. This changes the Biuret reagent from its pale blue color to violet or purple. The intensity of the color depends on the amount of Biuret reagent used and on the amount of protein in the sample.

Amino acid Dipeptide

peptide bond Polypeptide

Aspartame/Nutrasweet—a dipeptide

Lab 1 - 3 Procedure: Biuret Test

Controls:

What should you use as your negative control solution for this test? Obviously, something that is known to contain no polypeptides or free amino acids. One easily available negative control for this test is distilled water.

What should you use as your positive control solution for this test? Look over the available solutions and choose one you believe would be appropriate. a. Obtain 10 test tubes. Label the test tubes using a Sharpie pen to indicate the 10 solutions you will test (see Table 1-1, A1.) b. Add 3 ml of each test solution to the appropriate labeled test tube. See Table 1-1, A1.

If you have not used a transfer pipet before, ASK your instructor or TA to demonstrate!

It is very important that you not cross-contaminate the solutions. Be sure to use the correct pipet for each solution. If you accidentally contaminate the pipet with another substance, please ask your instructor for a clean pipet (they are disposable, so it’s not a big deal).

c. Add 3 ml of Biuret solution to each of the 10 test tubes and thoroughly mix by gently tapping the test tubes. d. The production of a violet color, a positive Biuret test, depends upon the presence of peptide bonds. In the presence of free amino acids, a pink-lavender color develops. e. In Table 1-1, A1, record whether your test solutions gave a positive or negative result and be sure to record the color/appearance of the solution.

Lab 1 - 4 B. CARBOHYDRATES Sugars and starches are examples of organic compounds known as carbohydrates because they contain carbon, hydrogen, and oxygen with a ratio of two hydrogens to each oxygen (with a chemical formula of (CH2O)n, where n can be any number). Carbohydrates are divided into several classes: the monosaccharides, the simple sugars, consisting of molecules with as few as three and as many as seven carbon atoms in the chain (e.g., glucose, which is a six-carbon molecule); the disaccharides, consisting of two simple sugar molecules linked together (e.g., table sugar, or sucrose, is made up of glucose linked to fructose); and the polysaccharides, which consist of many sugar molecules linked together (e.g., starch, glycogen, and cellulose). Fructose Glucose

HO HO OH HO OH

Monosaccharides serve as immediate sources of energy for cells; they are the only carbohydrates that can be absorbed directly by cells in the digestive tract. Disaccharides such as sucrose and lactose (milk sugar) must be broken down by enzymes into monosaccharides before they can be used for energy. Sucralose, a chemically modified sugar marketed as Splenda, cannot be broken down by enzymes and so passes undigested through the digestive system.

Sucrose Lactose Sucralose/Splenda

Starch and glycogen are both polysaccharides made from joining many, many glucose molecules together. These compounds serve as energy storage in many organisms (starch in plants and glycogen in animals) and must be broken down to monosaccharides by enzymes. Starch itself is made up of two different kinds of polymers of glucose: amylose (unbranched chains of glucose) and amylopectin (branched chains of glucose). Because of its branched structure, amylopectin is stickier than amylose. Different foods made from plants contain different amounts of these two types of starch. For example, sushi rice is sticky because it contains more amylopectin, while long grain rice is not sticky because it contains more amylose.

Lab 1 - 5 Amylopectin

Cellulose

Amylose

Glucose molecules

The enzyme amylase breaks down both amylopectin and amylose to glucose molecules that can be absorbed by cells in the digestive tract. Amylase is found in your saliva and your digestive tract (test this by holding some starchy food such as rice or an unsalted saltine cracker in your mouth for some period of time—the amylase in your saliva will change the starch into sugar! You need to be patient as this enzymatic digestion may take a few minutes.)

Cellulose is a polysaccharide similar in structure to starch; one small change in its structure makes it unable to be digested by most organisms (because the enzyme amylase cannot break it down). Cellulose is the most abundant organic molecule on earth and is found in plant cell walls where it provides important structural support.

Carbohydrates are readily identified by color reactions with specific reagents. These tests can be used quantitatively by describing the variation in color obtained with different carbohydrates. Monosaccharides, disaccharides, and polysaccharides require different testing procedures.

Lab 1 - 6 B1. MONOSACCHARIDES: BENEDICT'S SOLUTION TEST

Benedict’s solution contains copper ions (cupric ions, Cu++) that can be reduced by some sugars to form different copper ions (cuprous ions, Cu+) that form a precipitate in an alkaline solution. Depending on how much reducing sugar is present, the solution can turn yellow or green (for smaller amounts) or orange, red or brown (for larger amounts). This test is commonly done to test for glucose in human urine (an indicator of diabetes). For the protocol that we will use in lab today, monosaccharides will act as reducing sugars and should give a positive test result. Some disaccharides (depending on the details of their molecular structures) can reduce the copper ions; some cannot. Polysaccharides cannot reduce the copper ions and should give a negative test result. Controls: What should you use as your negative control solution for this test? Something that is known to contain no monosaccharides (or other carbohydrates to be safe). What should you use as your positive control solution for this test? Look over the available solutions and choose one you believe would be appropriate. Procedure: a. Obtain 14 test tubes and label them to indicate the ten solutions you will test (see Table 1-1, B1.) b. Add 10 drops of each test solution to the appropriate labeled test tube (see Table 1-1, B1.). c. Add 3 ml of Benedict's solution to each tube. Gently swirl the test tubes to be sure the contents are thoroughly mixed. d. Boil the test tubes cautiously for 2 minutes in a water bath. HANDLE HOT TEST TUBES WITH A TEST TUBE HOLDER. Remove tubes from the water bath and allow them to cool for 2 minutes (the colors may change over time, so it is important to record all your results at the same time). e. Record your results in Table 1-1, B1. B2 and B3…complete the questions related to amylase and artificial sweeteners that are on your worksheets.

B4. STARCH - I2KI TEST

Don’t forget to designate positive and negative controls for your I2KI test.

Be careful not to get I2KI on your skin or clothing; if you do, wash it off promptly.

To test for starch, add a few drops of iodine solution (iodine-potassium iodide, I2KI) to several drops of the substance you wish to test (see Table 1-1, B4) in a well of a porcelain plate. If there is starch present, the solution will turn bluish black as the starch and iodine form an adsorption complex. Record your observations in Table 1-1, B4.

Lab 1 - 7

C. LIPIDS

Lipids are a heterogeneous group of chemicals (fats, waxes, oils, steroids) that are classified together only because they are all relatively insoluble in water but are soluble in ether, acetone, and carbon tetrachloride. The simplest lipids, like the carbohydrates, are composed of carbon, hydrogen, and oxygen. Lipids are a major component of cell membranes. Fats serve as concentrated sources of energy and supply more than twice as many calories per gram as do carbohydrates and proteins. We will only test for fats today.

Fats are made up of three fatty acids linked to a glycerol molecule—they are thus called triglycerides. Fatty acids themselves are made up of two groups—a polar functional group and a nonpolar hydrocarbon chain. If the hydrocarbon chain contains all single bonds, it is said to be saturated. If the hydrocarbon chain contains one or more double C=C bond, it is said to be unsaturated.

Hydrocarbon chain of all single bonds

Polar functional group (carboxyl group)

Saturated fatty acid (stearic acid, especially common in animal fats)

double C=C bond causes kink in chain

Unsaturated fatty acid (oleic acid, especially common in olive oil)

Saturated triglyceride or “fat”, more solid

Lab 1 - 8 When saturated fatty acids are joined to make a saturated triglyceride or “fat,” the hydrocarbon chains are very close together and form a more solid fat. When unsaturated fatty acids are joined to make an unsaturated triglyceride, the double bond(s) cause kinks in the hydrocarbon chains, preventing them from packing close together—this makes for a less solid, or more liquid, fat (fats that are liquids at room temperature are called oils). Knowing this, can you guess whether butter or olive oil (both are made up primarily of triglycerides) contains more saturated hydrocarbon chains?

Because of the nonpolar nature of the hydrocarbon chains, fats are hydrophobic, that is, they don’t like water. If you mix olive oil and water (for example, when you make salad dressing), the oil and water will separate because the oil is hydrophobic. The same thing is true of fats in your body. Triglycerides stick together and exclude water. This is not true for carbohydrates (they are polar and like to have lots of water around; each pound of glycogen is associated with four pounds of water). It’s a good thing for us that our primary energy storage is in the form of triglycerides rather than glycogen. An average human might have enough glycogen to provide energy to last a day, but enough fat to provide energy for a month. If we didn’t have fat to store energy, we’d weigh an average of 80 lbs more (from the water that would associate with that huge increase in stored glycogen!). When you go on a diet, your body first uses its stores of glycogen. If you go on a crash diet, you will quickly use your stored glycogen and its associated water. Initial loss of weight is mostly due to water loss.

Trans Fat Unsaturated fats tend to be liquid at room temperature. In order to make them more solid and extend the shelf life of processed foods, many companies “hydrogenate” unsaturated fats to make them more saturated. The hydrogenation process not only adds hydrogens and removes C=C double bonds, it also converts some cis C=C double bonds into trans C=C double bonds, making trans-fats. Trans-fats are found in most processed foods, anything where the label says “partially hydrogenated” oil. It was thought for many years that hydrogenated fats (only somewhat saturated) were healthier than more saturated, naturally occurring fats. This is why many people switched from using butter (saturated animal fat) to using margarine (less saturated plant fat made by partially hydrogenating corn oil). However, as more research continues to be done, the evils of trans-fats are becoming evident. It may have an even greater contribution to heart disease than more saturated fat. While the jury is still out on which one is worse, it’s clear that both saturated fat and trans-fats contribute to heart disease, and you would do well to monitor your intake of both (be especially careful at the salad bar, studies show that the average American woman gets more fat from salad dressing than from any other single food). Labeling of trans-fats will be coming soon to nutritional labels (labeling will be required by Jan. 1, 2006).

Cis- double bond trans- double bond

Lab 1 - 9 Olestra is a fat substitute that is made from sucrose and 6-8 fatty acids. The enzymes that normally digest triglycerides in our digestive tracts break off the fatty acids from the glycerol molecules one at a time. Then, each fatty acid is digested. Olestra has at least twice as many fatty acids as triglycerides, so it should provide twice as many calories for our bodies. However, the enzyme (lipase) that remove the fatty acids from the glycerol can’t get into the Olestra to remove the fatty acids—the molecule is simply too bulky. Therefore, Olestra tastes like fats (because of its fatty acids), but can’t be digested like fats (because it is too bulky for lipase). It has effectively no calories because it passes through our digestive systems undigested. Before you eat a whole bag of potato chips fried in Olestra, you should check the warning on the back of the bag (bad things like “anal leakage” can happen when too much fat passes through the digestive system).

Olestra vs. Triglyceride (molecular models)

lipase enzyme cannot get in to release fatty acids from sucrose molecule in the lipase enzyme cuts here middle to release fatty acids from glycerol

Lab 1 - 10 1. SUDAN III DYE TEST FOR FATS

A specific test for fats is their selective uptake of the pigment Sudan III. This test is occasionally done on fecal samples to test for fat content (which can be an indicator of some diseases).

Don’t forget to designate your controls.

Using a graphite pencil, mark a filter paper disc with numbers corresponding to the test substances listed in Table 2-1, C2. Mark so that the numbers are equally spaced on the filter paper. Draw a small circle next to the letter.

a. Using a pipet, add a small drop from each tube to the appropriate circle on the filter paper. Return each pipet to its proper solution when you finish with it. b. Allow the paper to dry completely. c. Place the filter paper in a petri dish and add enough Sudan III solution to cover and soak through the paper. d. Soak the filter paper for 3 minutes in the Sudan III solution. e. Using forceps, remove the paper from the stain and rinse the paper with water (using the squeeze bottle over the sink!) for one minute. f. Examine the intensity of pink/orange staining of the 5 spots. g. Record your observations in Table 2-1, C2.

GENERAL END-OF-LAB PROCEDURES

Lab 1 - 11 You have now finished your first laboratory exercise. However, you are NOT yet ready to leave the lab. Before you go:

 Look over your data sheets before you leave lab. You will complete and turn in your data sheets to your instructor at the beginning of lab next week. If you have any questions, you are strongly encouraged to ask your instructor BEFORE YOU LEAVE LAB.

 Remember that there will soon be another section taught in your lab room. Please return everything to where you found it so that the next students can carry their labs out as efficiently as possible.

Clean up your lab bench:

o Return materials to where you got them from during lab.

o Dispose of any solutions down the drain (unless specified otherwise). Do not dispose of solids down the drain; please put them in the trash.

o Throw away any garbage you have generated.

o Place used glassware in labeled containers.

o Wipe off your lab bench and push in your chair.

 If you have any questions regarding the lab or other procedures, please feel free to ask your lab instructor or Margaret Olney or Karolin Rafalski in the Intro Biology Office (Altschul 911, 854-2153).

Lab 1 - 12 BIOLOGY 1001 NAME______Lab day and time ______Lab instructor______

DATA SHEETS: LABORATORY 1--Chemical Aspects of Life I. Complex Organic Molecules 1. Name the 4 classes of organic polymers that compose living matter. Name the monomers that make up each of these classes. Polymers Monomers

TABLE 1-1. RESULTS OF CHEMICAL TESTS A1. Biuret Test. Tests for what class of compounds?______

Positive control: ______Negative control: ______

Test Substance +/- Color Description tube A1.1 Distilled Water

tube A1.2 Egg Albumin

tube A1.3 Gelatin

tube A1.4 Blood Serum

tube A1.5 Skim Milk Fruit Juice (what kind? tube A1.6 ______) Aspartame/Nutrasweet in tube A1.7 Clear-Colored Diet Soda tube A1.8 Protein Solution

tube A1.9 Gatorade

tube A1.10 Unknown

Lab 1 - 13 TABLE 1-1. RESULTS OF CHEMICAL TESTS – Continued

B1. Benedict’s Test. Tests for what class of compounds?______

Test Substance +/- Color tube B1.1 Distilled Water tube B1.2 5% Glucose tube B1.3 5% Sucrose tube B1.4 Whole Milk tube B1.5 Skim Milk tube B1.6 Lactaid Milk tube B1.7 Soy Milk tube B1.8 Blood Serum Fruit Juice (specify what kind tube B1.9 ______) tube B1.10 Potato Juice tube B1.11 Clear-Colored Non-Diet Soda tube B1.12 Clear-Colored Diet Soda tube B1.13 1 % Cornstarch tube B1.14 Unknown

B2. Amylase enzyme activity test a. Hold an unsalted saltine in your mouth. How long does it take to begin to taste sweet?

______minutes b. Describe the enzymatic reaction taking place inside your mouth that is making the cracker taste sweet.

B3. Artificial sweetener taste test

Taste the table sugar (sucrose), Nutrasweet (aspartame), and Splenda (sucralose) samples. a. Rank them in order of least sweet to most sweet.

b. Are there any differences in taste that you can detect?

c. Briefly describe how the structures of aspartame and sucralose differ from sucrose.

d. Check the side of the box to see how the calorie content of aspartame and sucralose compare to those of sucrose.

e. Why do aspartame and sucralose have fewer calories than sucrose? (Hint: think about enzymes.)

B4. I2KI Test. Tests for what class of compounds?______

Test Substance +/- Color tube B4.1 Distilled Water tube B4.2 1% Starch Solution tube B4.3 Soy Milk

Fruit Juice (what kind? tube B4.4 ______) tube B4.5 Potato Juice tube B4.6 Unknown

C. Sudan III Test. Tests for what class of compounds? ______

Test Substance +/- Intensity of Pink Color spot C.1 Distilled Water Vegetable Oil I (what kind? spot C.2 ______) Vegetable Oil II (what kind? spot C.3 ______) spot C.3 Skim Milk spot C.4 Whole Milk spot C.5 Blood Serum spot C.6 Soy Milk spot C.7 regular fatty potato chip spot C.8 Olestra fried potato chip tube C.9 Unknown

Lab 1 discussion questions:

1. Into what class of compounds would you place your unknown? Why?

Lab 1 - 17 2. Describe any surprises in your results of the various tests. Try to explain them.

3. You were having a dinner party for 17 of your closest friends. You made two kinds of salad dressing for them to try. For one of the dressings, you used olive oil; for the other, soybean oil. You placed the dressings in the refrigerator to chill before the party. When it came time to serve the salad, you removed the two bottles from the fridge. To your surprise, the olive oil had solidified and could not be served to your guests (it was impossible to shake to mix!), but the soybean oil dressing remained liquid. Explain the structural difference in the chemistry between olive oil and soybean oil that brought about this distressing situation. You may wish to include a labeled diagram to accompany your answer.

4. Compare the tastes of regular and Wow potato chips? Can you detect a difference in flavor or crispiness? Check the nutrition label and compare calorie contents. Can you explain the difference in calories?

Lab 1 - 18