Proteins, Carbohydrates, and Lipids

By: Addie Lupercio, Andrea Elguezabal, Akido Sheard, and Daniel Attwood

Introduction
o Macromolecules- made of many single monomers held together through covalent bonds o Lipids- They are not actually macromolecules because they're not made up of monomers but they are built through covalent bonds called ester linkages. Lipids are grouped together because of their hydrophobic nature and are made up of glycerol and usually 3 fatty acids. One common exception is phospholipids, their main purposes are energy storage, cushion, and insulation. We test for them by seeing if they will evaporate from paper or leave an oily spot. The size of the spot after 5 min shows the amount of lipids in the substance.

o Carbohydrates-Made from monosaccharides such as glucose. They are held together by glycosidic linkages in either alpha or beta ring structures to form disaccharides, polysaccharides and starches. Their primary uses are for storage and building materials. We test for starches with iodine which will turn a blue-black color and for different concentrations of sugars using Benedict's reagent after heating. o Proteins- Made from amino acids held together through peptide bonds. Amino acids consist of an amino group, a carboxyl group, and a variable side chain called the R group. Amino acid chains form polypeptides which in turn fold and coil into proteins. The 3-D structure of proteins is very complex and specific to its function. The test using the Biuret reagent, reacts to the amino group in amino acids producing a pinkish color. However due to the quaternary structures of proteins this makes fewer amino groups available.

Materials and Methods

Protein Experiment 1. The first test was on proteins. We had 3 test tubes. In the first one we added 2ml of distilled water. 10-15 drops of Biuret reagent were added to the distilled water and mixed gently by flicking the lower portion of the test tube. Then we recorded the color change in Table 1.1. This was used as our negative control. 2. To the second test tube we added 2ml of 1% albumin solution. 10-15 drops of Biuret reagent were added to the albumin solution and gently mixed. The final color was then recorded in Table 1.1. This was our positive control. 3. Test tube 3 was filled with 2ml of the test substance. 10-15 drops of Biuret reagent were added and gently mixed with the test substance. The color change was then recorded in Table 1.1. This was our experimental tube.

Starch Experiment

1. The next test was on starch. We used three test tubes for this experiment. The first test tube was filled with 1ml of distilled water. Then two to five drops of iodine solution were added to the distilled water. The final color was recorded in Table 2.1. 2. In test tube number two we added 1ml of 1% starch suspension that was shaken very thoroughly prior to putting into the test tube. Two to five drops of iodine solution were added to the starch suspension. The final color change was then recorded in Table 2.1. 3. To test tube three we added 1ml of the test substance. Two to five drops of iodine solution were added to the test substance. The final color change was recorded in Table 2.1.

Sugar Experiment

1. The third test was for sugars. Three test tubes were used for this experiment. In the first tube 1 ml of distilled water was added. 2 ml of Benedicts reagent was added to the distilled water. This was then placed into a boiling water bath for 5 minutes. Noting any of the changes that occurred in the five minutes in Table 3.1 This was the negative control for this experiment.

2. In test tube two 1 ml of 1% glucose solution was added. 2 ml of Benedicts reagent was added to the glucose solution. This was then heated in a boiling water bath for 5 minutes. We then noted any changes that took place, during those 5 minutes, in Table 3.1.
3. In test tube three 1 ml of the test solution is added. 2 ml of Benedicts reagent is added to the test solution. This is placed into a heated water bath for 5 minutes. Noting any color changes that occurred during the 5 minutes in Table 3.1.

Lipid Experiment

1. Test number four was on lipids. We put on gloves for protection. We used a large piece of brown paper. We then placed a small drop of water onto the brown paper. With our gloved finger we rubbed that water into the paper. 2. Second, we placed a small drop of vegetable oil onto the brown paper and with our gloved finger and rubbed it into the brown paper. 3. Lastly, we dropped a small amount of the test solution onto the brown paper. Using our gloved finger we rubbed the test solution into the brown paper. We then waited 5 minutes to allow for the three solutions to evaporate. We then recorded the results in Table 4.

Results
Table 1. Test for protein.

Proteins

CHEMICAL
Biuret reagent (blue)

PROTEINS (complex)
Violet

PEPTIDES (simple)
Pinkish

Table 1.1 Different test substances mixed with a Biuret reagent solution in order to determine the
presence of proteins and peptides.

TUBE CONTENTS
1 2 3 Distilled Water Albumin Test Substance

FINAL COLOR
Light Blue/Clear Purple Red-orange/Pinkish

CONCLUSIONS
There were no proteins or simple peptides in this solution. There were high levels of protein in this solution. There were high levels of simple peptides in this solution.

When the Biuret reagent (blue in color) was mixed with the distilled water in test tube one it did not change in color, it was merely diluted, as table 1 shows its color was light blue. The results for this test tube showed there werent any proteins or simple peptides in this solution. The second test tube did demonstrate a color change to purple. This test tube contained Albumin, a substance made of proteins which have a complex structure. The last test tube turned a red-orange/pinkish color when mixed with the Biuret reagent. This gave evidence that the test substance did contain simple peptides in the test substance.

Starch
Table 2. Test for Starch
CHEMICAL COLOR COLOR (with Starch)
Blue-Black Potassium Iodine Brownish

Table 2.1 Different solutions mixed with Iodine in order to test for the presence of starch.
TUBE CONTENTS
1 2 3 Distilled Water Starch Test Substance

COLOR CHANGE
No color change Blue-black/Dark Purple No color change

CONCLUSION
The solution does not contain starch. Starch is present in the solution. The solution does not contain starch.

The first test tube, our negative control, had no change in color as there was no starch present in the distilled water. The second test tube, our positive control, did react with the Iodine as it was a starch solution. The third test tube, our test substance, demonstrated no color change. The solution did not contain starch and therefore did not have a reaction with the Iodine.

Sugar
Table 3. Test for Monosaccharides.
CHEMICAL CHEMICAL CATEGORY Distilled Water Inorganic Glucose Monosaccharide (carbohydrate) BENEDICT'S REAGENT (after heating)

Blue (no change) Varies with concentration: Very low - green Low - yellow Moderate - yellow-orange High orange Very high orange-red

Table 3.1 Different solutions mixed with Benedicts Reagent in order to detect the presence of
monosaccharides.

TUBE CONTENTS COLOR (After Heating)

1 2 3 Distilled Water Blue Glucose Solution Orange-red/Dark Red

CONCLUSIONS
There were no monosaccharides in this solution. There were very high levels of monosaccharides in this solution. There were moderate levels of monosaccharides in this solution.

Test Substance Yellow/Orange

The first test tube, our negative control, did not change in color as there werent any monosaccharides in the distilled water. The second test tube, our positive control, changed color to an orange-red/Dark Red. This change in color showed the high levels of monosaccharides in the solution. The third test tube, our test substance, demonstrated a change in color to a yellow/orange color. The color of this solution gives us evidence that there are moderate levels of monosaccharides in this solution.

Lipids

Table 4. Paper test for lipids.

SAMPLE
Distilled Water Vegetable Oil Test Substance

RESULTS
The water evaporated off of the paper. The paper remained wet/oily. There remained some traces of oil on the paper.

The water, our negative control, evaporated from the paper. This demonstrated that there were no lipids in the water solution.
The vegetable oil, our positive control, left a wet/oily spot on the paper. This shows evidence that there were lipids present in the vegetable oil. The third sample, our test sample, left some traces of oil on the paper. This showed us that there were some lipids in the solution.

Discussion
Due to the fact that all of our experiments were on unknown substances we did not form any hypotheses. We instead focused on establishing our positive and negative controls to ensure our results were valid. We were able to establish the varying percentages of macromolecule within the unknown test substances. As far as we could tell there were no variations or unexpected results. In future experiments we could select several known substances and test them for the varying amounts of each macromolecule within them.

Conclusion
In conclusion,
The test substance was negative for proteins. Albumin was used as a positive control. The test substance was also negative for carbohydrates. Iodine solution turns a brownish color in the presence of starch because the iodine-starch complex absorbs most visible wavelengths of light. The test substance showed no change. The test substance showed positive for sugars, although it was moderate. When added to benedicts reagent, after heating, the test substance yielded a yellow/orange color. The positive control, glucose solution, turned red/orange. The negative control, distilled water, showed no change.

We were not able to deduct what the test substance was from these results.

References
Text Campbell Biology, Reece et al. 2011 Test Videos http://www.youtube.com/watch?v=OLxJTuvei90 - Biuret test for protein and peptides http://www.youtube.com/watch?v=Lt7RCIfudYQ - benedict test reducing sugars http://www.youtube.com/watch?v=HO_q8GPl3bE - iodine test for starch