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Laboratory 3 – Organic Molecules MATERIALS

• Distilled water, vegetable oil, and solutions of glucose, starch, and gelatin • Dairy products (half and half, heavy cream and whole milk) • Non-dairy soy and almond milk • The biuret reagent and 1% NaOH • IKI (iodine potassium iodide or Lugol’s iodine solution) • Benedict’s reagent • Sudan IV/glycerol solution • 70% ethanol • White ceramic spot plates, china markers (grease pencils), toothpicks, and eyedroppers • Pipettes, test tubes, test tube racks, and test tube holders • Thin tissue sections cut from beans, potatoes, and almonds • Tweezers, microscope slides, cover slips, and a No. 2 pencil • 100 °C water bath, 4 °C ice bath, and vortex mixer

LEARNING OUTCOMES Upon completion of the exercises in this laboratory, you should be able to: 1. Use the appropriate biochemical tests to determine the presence of three types of organic

molecules 2. Distinguish the positive from negative outcomes for each biochemical test 3. Establish a hypothesis 4. Collect and interpret results 5. Identify the specific type of milk being tested based upon the organic molecules it contains Living organisms are composed of four large groups of organic molecules: carbohydrates, proteins, lipids, and nucleic acids. Each has a specific biologic role, and very often a specific distribution within a living organism. A knowledge of an organic molecule’s chemical properties, functions, and interactions are fundamental to understanding cellular processes. Therefore, the detection of organic molecules in laboratory tests is a necessary part of the study of organisms. Specific biochemical tests have been developed to detect organic molecules, some of which will be used in this and future laboratory exercises. The identification of organic molecules is also useful in the analyses of the composition of foods derived from both plants and animals. The organic molecules found in foods vary depending on their source and post-harvest processing. Many food products are modified from their native states to satisfy the needs of consumers. For example, lactose intolerant individuals cannot process lactose (a disaccharide of galactose and glucose) leading to problems in their digestive tracts. Manufacturers of dairy products market ‘lactose free’ milk to reduce these problems. Manufacturers also adjust the amount of lipids in milk so as to range from ‘fat free’ (0% lipid) to ‘whole’ (~3.2% lipid). Confusingly, several plant-based (non-dairy) ‘milks’ are also available to consumers including almond and soy milk. We can compare the organic molecules of dairy vs. non-dairy ‘milks.’ In this laboratory, several ‘milks’ and other solutions will be tested to identity their constituent organic molecules.

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Appropriate laboratory techniques and procedures must be followed for accurate results. Scientific procedures, similar to cooking recipes, require care and precision for a successful outcome. In addition, laboratory tests must include both positive and negative controls to establish their validity. To identify organic molecules, a positive control illustrates a specific (positive) result with a known molecule. A negative control will lack any molecules that will generate this positive result. In our experiments, a negative control often substitutes water for other molecules. A positive reaction will involve a visible change in the color of a solution inferring that a particular organic molecule is present within it. The controls thus establish reference points for all the reactions performed on solutions with unknown compositions. Separate tests will be performed to detect the presence of proteins, carbohydrates, or lipids. Questions

1. Does milk processing influence its constituent organic molecules?

2. Do non-dairy and dairy milk contain the same organic molecules?

3. Can specific tests performed in this laboratory distinguish between dairy products? The samples to be analyzed include whole milk, heavy cream, half and half, almond milk, and soy milk. Based upon previous knowledge, establish a hypothesis for each question above and provide a rationale for it: Hypotheses

1.

2.

3.

Rationales

1.

2.

3.

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Note that each specific test will require a positive and negative control. Water and separate solutions containing glucose (a simple sugar or monosaccharide), starch (a polymer sugar or polysaccharide), gelatin (a mixture of proteins, mainly collagen) and vegetable oil (lipids) will be provided. Exercise 3.1 – Detection of Lipids (Sudan IV Test)

Lipids (fats and oils) are very prevalent components of most organisms. Lipid molecules, due to their non-polar bonds, are hydrophobic (lipophilic) and as such do not readily mix with water. They are soluble in various organic solvents including ethanol, hexane, and ether. Phospholipids and cholesterol (also a lipid) are major components of many cell membranes. Cholesterol tends to stiffen most cellular membranes. Cholesterol is also the parent component for the production of steroids (i.e.; testosterone, estrogen, cortisol). The major fats of both animals and plants, in structures known as triglycerides, are used to store energy. Curiously, hydrophobic lipids are commonly found within watery cells or the watery fluid that surrounds cells. One would expect the lipids to be separated from the watery environments due to their hydrophobicity. Using internet resources, try to use relevant information to establish a hypothesis about how lipids are maintained within these hydrophilic environments. Hypothesis

Most biochemical tests to detect lipids rely upon lipophilic (lipid soluble) reagents. Sudan IV* is a lipophilic substance that when mixed with lipids stains them red.

Figure 3.1 – Sudan IV (A Fat-Soluble Dye)

* Michaelis, L. (1901). Uber fett färbstoffe [Dyes for fats]. Virchow Arch 164:263-270.

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Procedure 3.1 – Detecting Lipids with Sudan IV

1. Obtain an appropriate number of test tubes and place them in a rack on your laboratory

bench. Label the tubes with a china marker (grease pencil).

2. Add 20 drops (1 ml) of distilled water into one test tube, and the same amount of each

solution listed in Table 3.1 to a separate, appropriately labeled test tube. Use a separate

pipette for each solution.

3. Add 20 drops (1 ml) of distilled water into each tube listed in Table 3.1 and gently mix

the contents with a vortex mixer for about 5 seconds.

4. Add 10 drops of the Sudan IV/glycerol solution to each test tube and gently mix the

contents with a vortex mixer for about 5 seconds. Pipette the Sudan IV into your tubes at

the designated lab bench and do not bring the stock solution of Sudan IV to your bench.

5. Allow the tubes to remain within an undisturbed rack for 5 minutes. It may be necessary

to place the tubes in an ice bucket for 15 – 30 minutes for clearer results.

6. The appearance of a red layer within the solution implies the presence of lipids. Use the

negative control for comparison and record the visual results in Table 3.1. Note any

difference in the intensity of the red color. Observe the results again after the incubation

of the tubes on ice.

7. Discard the contents of the test tubes in a designated waste container, wash them

thoroughly with soap and tap water, rinse them extensively with tap water, and place

them inverted in a test tube rack to dry upon your laboratory bench.

Table 3.1 – Detection of Lipids (Sudan IV Test) Results

Tube # Solution Final Color Presence (+) or Absence (–) of Lipids

1 Water 2 Glucose 3 Starch 4 Gelatin 5 Vegetable oil 6 Milk A 7 Milk B 8 Milk C 9 Milk D 10 Milk E

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Exercise 3.2 – Detection of Proteins

Proteins are polymers of specific arrangements of differing amounts of 20 amino acids linked together by peptide bonds. An enormous number of proteins can be synthesized through different combinations and numbers of amino acids. This diversity leads to numerous cellular functions for proteins including structural (e.g. within the cytoskeleton, and extracellular matrix) and functional (e.g. enzymatic, hormonal, and secretory) roles. Peptide bonds are a specific chemical feature in proteins that separates them from other organic molecules. They are formed by a dehydration synthesis (polymerization reaction) that generates a covalent bond between two amino acids. The peptide bond (marked with a * in Figure 3.2) within a protein is specifically found between the carbonyl (C=O) group on one amino acid and the amine group (N-H) of another. *

Figure 3.2 – Peptide Bond Formation A specific test to detect proteins is known as the biuret reaction* in which a blue solution containing copper sulfate (CuSO4) changes to a violet color in the presence of two or more peptide bonds. A simplification of the reaction is illustrated in Figure 3.3.

Figure 3.3 – The Biuret Reaction The solutions listed in Table 3.2 will be tested for the presence of proteins utilizing the biuret reaction.

* Rose, F. (1833). Ueber die verbindungen des eiweiss mit metalloxyden [On the compounds of proteins with metal

oxides]. Annalen der Physick 104: 132-142.

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Procedure 3.2 – Detecting Proteins (Biuret Reaction)

1. Obtain a white ceramic spot plate, the biuret reagent, eyedroppers, and the solutions

listed in Table 3.2. Number the wells of the plate with a china marker (grease pencil).

2. With a supplied eyedropper, add 5 drops of 1% NaOH to each of the 10 wells.

3. With the supplied eyedroppers, add 5 drops of distilled water in one well, and 5 drops of

each organic solution listed in Table 3.2 to separate appropriately labeled wells. Use a

separate eyedropper for each solution.

4. Briefly mix the contents of each well with a separate and unused toothpick.

5. Add 3 drops of the biuret reagent into each well of the spot plate and mix the contents

with a toothpick. Use a separate toothpick for each individual well.

6. After mixing, the appearance of violet color in a well implies the presence of protein. Use

the negative control for comparison and record the visual results in Table 3.2.

7. Discard the contents of all wells of the spot plate in a sink, rinse the plate with tap water,

and dry it with a paper towel. Use the cleaned plate in the next exercise.

Table 3.2 – Biuret Reaction Results

Well # Solution Final Color Presence (+) or Absence (-) of Protein

1 Water 2 Glucose 3 Starch 4 Gelatin 5 Vegetable oil 6 Milk A 7 Milk B 8 Milk C 9 Milk D 10 Milk E

Review

What were your negative and positive controls and what were their functions in the experiment?

The biuret test is specific for proteins because:

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Exercise 3.3 – Detection of Starch (IKI Test)

Starch represents one of several polysaccharides composed of a single type of monosaccharide (simple sugar) known as glucose. Glucose monomers (single molecules) are the main source of energy for most cells. Cells store excess glucose by linking it together via glycosidic bonds (a type of covalent bond) forming polymers (chains of linked monomers) known as plant starch or animal glycogen. Saccharides and polymers of saccharides are collectively known as carbohydrates. Structurally, the starch molecule is a coiled and spring-like polymer of glucose. To detect starch in a substance one can add iodine (IKI or Lugol’s iodine solution) to it. Iodine molecules can associate with and accumulate within coils of starch*. Solutions of free iodine are dark amber (yellow-brown) in color. When iodine is concentrated within coils, a blue-black color develops, implying the presence of starch (see Figure 3.4).

Figure 3.4 – Iodine (purple spheres) Accumulating in Starch (connected white hexagons) Procedure 3.3 – Detecting Starch (IKI Test)

1. Use the cleaned white spot plate from the previous exercise. Determine how many wells

will be needed and label them with a china marker (grease pencil) appropriately.

2. With the supplied eyedroppers, add 5 drops of water in one well, and 5 drops of each

solution listed in Table 3.3 to separate appropriately labeled wells. Use a separate

eyedropper for each solution.

3. Add 1 drop of the IKI solution into each of the wells of the spot plate and mix the

contents with a toothpick. Use a separate toothpick for each solution.

4. The appearance of a blue-black color indicates the presence of starch. Use the negative

control for comparison and record the visual results in Table 3.3.

5. Discard the contents of the spot plate in a sink, rinse it with tap water, and dry it with a

paper towel.

6. Test for the presence of starch in different papers (e.g. towel paper, chromatography

paper, or this lab’s paper) by placing a drop of IKI on it:

Place 1 drop of IKI within this circle à

* Stromeyer, F. (1815) Ein sehr empfindliches reagens für iodine aufgefunden in der Stärke (Amidon) [A very sensitive reagent for iodine found in starch]. Annalen der Physick 49: 146–153.

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Table 3.3 – IKI Test Results

Well # Solution Final Color Presence (+) or Absence (–) of Starch

1 Water 2 Glucose 3 Starch 4 Gelatin 5 Vegetable oil 6 Milk A 7 Milk B 8 Milk C 9 Milk D 10 Milk E

Review

Starch is a polysaccharide carbohydrate. Will IKI react with all carbohydrates? Explain.

Which numbered well can be used to explain the answer to the previous question? Why?

During manufacturing starch is often mixed in with the wood pulp (cellulose fibers) to strengthen paper. How do you think starch does this?

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Exercise 3.4 – Detection of Reducing Carbohydrates (Benedict’s Test)

The building blocks of di- and polysaccharides are the so-called simple sugar monosaccharides (i.e.; glucose, fructose, and galactose). In the body, dietary monosaccharides are absorbed without modification and are later converted to glucose within the liver. In addition, dietary carbohydrates include di- and polysaccharides that are enzymatically digested to monosaccharides before cellular absorption. The common disaccharides include maltose (glucose–glucose), sucrose (glucose–fructose) and lactose (galactose–glucose). Monosaccharides and some disaccharides (known as reducing sugars) can be detected by a test utilizing another copper-based solution known as Benedict’s reagent.* This method relies upon the ability of reactive (reducing) mono- and disaccharides to donate electrons to Cu++ ions of Benedict’s reagent. In solution, when Cu++ gains a negative electron it becomes (reduces to) Cu+ and a color change from blue to brick-red is observed. Specifically, the color of a positive test can range from green (low sugar concentration), to orange-brown (moderate sugar concentration), and to brick red (high sugar concentration). A simplified version of the reaction is illustrated in Figure 3.5.

reducing sugar Benedict’s reagent (blue) oxidized sugar reduced Benedict’s reagent (red)

Figure 3.5 – Reduction of Benedict’s Reagent by a Sugar Molecule Procedure 3.4 – Detecting Reducing Sugars (Benedict’s Test)

1. Obtain an appropriate number of test tubes and place them in a rack on your laboratory

bench. Label the tubes with a china marker (grease pencil).

2. Pipette 1 ml of water into one test tube, and the same amount of each solution listed in

Table 3.4 to separate and appropriately labeled test tubes. Use a separate eyedropper for

each solution.

3. Pipette 1 ml of the Benedict’s reagent to each test tube and gently mix the contents with

a vortex mixer for about 5 seconds.

4. Place all the test tubes within a 100 °C water bath for 2 to 5 minutes.

5. Carefully remove the hot tubes individually from the water bath using a test tube holder,

place them in a test tube rack, and bring them back to your laboratory bench.

* Benedict, S.R. (1909). A reagent for the detection of reducing sugars. Journal of Biological Chemistry 5: 485-487.

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6. The appearance of a green, orange, or brick-red solution implies the presence of reactive

(reducing) mono- or disaccharides. Use the negative control for comparison and record

the observed results in Table 3.4. Note that each specific color corresponds to an overall

concentration of reducing mono- and disaccharides within each solution.

7. Discard the contents of the test tubes in a sink, wash them thoroughly with soap and tap

water, rinse them extensively with tap water, and place them inverted in a test tube rack

to dry upon your laboratory bench.

Table 3.4 – Detection of Reducing Sugars (Benedict’s Test) Results

Tube # Solution Final Color Presence (+) or Absence (-) of Reducing Sugar

1 Water 2 Glucose 3 Starch 4 Gelatin 5 Vegetable oil 6 Milk A 7 Milk B 8 Milk C 9 Milk D 10 Milk E

Review

The Benedict’s test detects glucose, the building block of starch, yet it does not detect starch. Explain.

Starch is consumed while eating french fries. What process can generate glucose molecules from this starch? Which types of molecules carry out that process?

What human disease could be monitored by Benedict’s test? What might be the easiest way to do this?

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Summary of Organic Molecules Detected Within Milks

Based upon the tests performed, and the data recorded in Tables 3.1–3.4, determine the content of the milks listed in Table 3.5. Try to identify the type of milk (i.e.; whole milk, heavy cream, half and half, almond, or soy) of each based upon the organic molecules found within them. Table 3.5 – Organic Molecules Detected within Milks Results

Milk Biuret

Reaction (+/–)

IKI Test (+/–)

Benedict’s Test (+/–)

Sudan IV Test (+/-)

Organic Molecules Detected

Sample Identity

Milk A Milk B Milk C Milk D Milk E Do your results support, or refute, each of the three hypotheses you proposed at the beginning of this laboratory? Explain your conclusions.

1.

2.

3.

Which of the milks tested will probably provide the most diverse nutrition?

Humans performing physically demanding activities require significant amounts of proteins and carbohydrates. If only vegetables are available, what could a menu for these individuals be supplemented with?

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Exercise 3.5 – The Detection of Organic Molecules Within Tissues

Organic molecules can be detected within specific areas of cells, or within specific tissues, using the reagents of the previous exercises. Color changes (staining) can be microscopically (histologically) observed within cells or tissues implying the presence and location of specific organic molecules. In a prior laboratory, a prepared permanent microscopic sample containing Paramecium were observed. In addition to permanent slides, slides known as wet mounts can be prepared from fresh living tissues. Wet mounts are usually intended for immediate observation and cannot be preserved for longer periods of time. The preparation of a wet mount is relatively simple. A thin tissue section is placed on a microscope slide, and a drop of water, or staining reagent, is added over the specimen. A small coverslip is then slowly lowered from a 45o angle over the sample (see Figure 3.6). Excess water and/or staining reagents that leaks outside of the edges of the coverslip can be gently wicked away with the edge of a paper towel.

Figure 3.6 – Preparation of a Wet Mount Slide

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Preparation and Staining of Living Plant Tissues Wet-mounts will be prepared from potato (Solanum tuberosum), white beans (Phaseolus vulgaris), and almond (Prunus dulcis) tissues. Each will be stained separately with an IKI solution, the Biuret reagent, and Sudan IV. After the staining procedures, coverslips are discarded as glass trash, but slides can be rinsed free of tissues with tap water and reused. Procedure 3.5 – IKI Staining

1. Obtain 3 clean microscope slides and label them within their frosted areas with a pencil.

2. Thin sections of each sample will be cut from each plant (your instructor will describe the

procedure and provide the sections). Add 1 drop of distilled water to each section laid

upon the center of a slide. Slowly lower a coverslip from a 45o angle over the section to

complete the wet mount. Remove any excess fluid with a paper towel before placing the

slide on the microscope stage.

3. Observe the wet mount under the microscope with scanning (40X), low (100X), and then

high power (400X) magnification. Sketch each tissue observed at 400X in Figure 3.7.

4. Carefully add 1 drop of IKI to one edge of the coverslip. It should mix with the water

under the coverslip, but the stain can also be drawn under by briefly applying a paper

towel to the opposing edge of the coverslip. Observe this wet mount under the

microscope as in the previous step. Draw your observations in Figure 3.8.

Procedure 3.6 – Biuret Staining

1. You will be provided with thin sections from potato, bean, and almond tissues.

2. Transfer the sections with tweezers into separate wells of a white spot plate and then add

2 drops of NaOH to each and incubate for 5 minutes.

3. Add 1 drop of CuSO4 to each well and gently agitate the plate with a circular motion on

the bench.

4. Transfer the sections onto slides with tweezers and then generate wet mounts for

observation under the microscope. Draw your observations in Figure 3.9.

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Procedure 3.7 – Sudan IV Staining

1. You will be provided with thin sections from potato, bean, and almond tissues.

2. Transfer the sections with tweezers into separate wells of a white spot plate and cover

each with 3 drops of the Sudan IV/glycerol solution. Allow the sections to stain for about

5 minutes.

3. Transfer the sections independently to other wells within the spot plate with tweezers and

then cover with 10 drops of 70% ethanol. Gently agitate the plate for about 30 seconds.

This step washes away excess Sudan IV.

4. Transfer the sections independently to a third set of wells of the spot plate with tweezers

and then cover with 10 drops of distilled water as a final rinse.

5. Transfer the sections onto slides with tweezers and then make wet mounts for observation

under the microscope. Draw your observations in Figure 3.10.

Potato Bean Almond

Figure 3.7 – Unstained Tissue Sections at 400X

Figure 3.8 – IKI Stained Tissue Sections at 400X

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Potato Bean Almond

Figure 3.9 – Biuret Stained Tissue Sections at 400X

Figure 3.10 – Sudan IV Stained Tissue Sections at 400X

Review

Which of the tissues stained positively for starch? For proteins? For lipids?

Can you localize any of the organic molecules to specific organelles within the cells you

observed? If so, what organelles stained positively for starch, proteins, or lipids?