Experimenting with Biological Macromolecules in Various Identification Tests

INTRODUCTION Macromolecules are highly important organized organic molecules that are found within living cells. These biological molecules help the body execute precise important tasks. There are four types of macromolecules: carbohydrates, lipids, proteins and nucleic acids. (Karp, 2010) This specific experiment showcased the identification of different macromolecules using three different testing methods.
Carbohydrates, sugar molecules, are known as “fast fuel” in the body, and they contain base elements of carbon, oxygen and hydrogen. (Lea, 2002) The simplest carbohydrate molecule is known as a monosaccharide, which is a simple sugar. Monosaccharide’s can be joined together by a covalent glycosidic bond to create more complex sugars such as disaccharides (2 monosaccharide’s), oligosaccharides (between three to ten monosaccharide’s) and polysaccharides (consists of several monosaccharide’s). (Karp, 2010) Starch is the most common nutritional polysaccharide, which consists of two polymers: amylose and amylopectin. Amylose is unbranched whereas amylopectin is branched. Structural polysaccharides consist of cellulose, which is a major component of plant cell walls; chitin, which is a tough outer covering of invertebrates; and glycosaminoglycans (GAGs). (Karp, 2010) Lipids help produce energy within the body. Lipids are hydrophobic (“water-hating”), which makes it harder to break down into energy for the body. Steroids, fats and phospholipids are different functional forms of lipids. Steroid molecules are created around a four-ringed hydrocarbon skeleton and provide aid in the synthesis of many steroid hormones. (Karp, 2010) Triglycerides are composed of three fatty acid molecules and one glycerol molecule that is bonded with an ester linkage. Fats that are liquid at room temperature are classified as being an oil. (Karp, 2010) Triglycerides and phospholipids both share a glycerol molecule. Their major difference is that the phospholipids only contain two fatty acids, instead of three, and they also contain a phosphate group that is covalently bonded to a polar head group. (Karp, 2010) Proteins are the macromolecules that carry out the most functions. Some of proteins complex functions include: acting as enzymes, hormones, antibodies, toxins, light absorption and refraction, transporting substances from one area of the body to another and a few more important functions. (Karp, 2010) Amino acids are the monomers of proteins and are bonded together in unique combinations to create polypeptide chains and finally the chain is folded into a “unique functional protein”. (Lea, 2002) The whole process of protein formation occurs during DNA transcription and translation by RNA. (Lea, 2002) Nucleic acids are composed of long strands of monomers called nucleotides. Nucleotides consist of three important parts: a five-carbon sugar (pentose sugar), a phosphate group and a nitrogenous base. There are two types of nucleic acids found amongst living organisms, double-stranded deoxyribonucleic acid (DNA) and single-stranded ribonucleic acid (RNA). Deoxyribonucleic acids provide all the genetic information of all cells whereas ribonucleic acid reads the information and helps carry out the messages of DNA. (Karp, 2010) For each of the three testing methods, the Iodine Test, Benedict’s Test and the Biuret Test, distilled water acted as the negative control and helped prove which sample solutions were indeed positive or negative.
The Iodine Test for starch and glycogen was the first testing method used in this experiment. Iodine solution has a base colour of pale yellow without contact with other solutions. Distilled water, being the negative control, showcased that the resulting colour of the solution would have to resemble a dark yellow liquid. A positive result for the iodine test was presented with the resulting colour change of the sample solution being violet-blue-black. (Gurien, 2008)
Secondly, Benedict’s Test for reducing sugars was performed. Reducing sugars means that the macromolecule has a sugar with a free aldehyde or ketone group. Benedict’s solution contains cupric ions, which attach themselves to a reducing sugar’s free aldehyde group. (Gurien, 2008) The negative control was determined to have a light blue resulting colour change. In this experiment, the samples that tested positive for the presence of a reducing sugar had an orange-red-brown colour change with some precipitate formed at the bottom of the test tube. (Gurien, 2008)
Lastly, the Biuret test was conducted to identify the presence of proteins in the sample solution. The Biuret solution is made up of sodium hydroxide and copper sulfate. In the presence of a protein the biuret reagent will turn violet, but if the sample is negative; based upon distilled water being the negative control; then the sample will stay a light blue. (Gurien, 2008)

MATERIALS AND METHODS

This lab was performed exactly like (Department of Biology 2001 Introductory Cell Biology Laboratory Manual. University of Waterloo, Waterloo. pp. 17-20) with no deviations

RESULTS Table 1.1: Iodine Test Results
The Iodine Test was preformed to find the presence of starch and glycogen in the 11 known sample solutions and the one unknown sample solution. The positive result for the existence of starch and glycogen in the samples was the colour change of violet-blue-black.
SAMPLE SOLUTIONS COLOUR CHANGE RESULTS POSITIVE/NEGATIVE
#1- 1% Glucose Solution Dark Yellow Colour Change Negative Result
#2 – 0.3% Glucose-1-Phosphate Dark Yellow Colour Change Negative Result
#3 – 1% Maltose Solution Dark Yellow Colour Change Negative Result
#4 – Honey Solution Dark Yellow Colour Change Negative Result
#5 – Sucrose Solution Dark Yellow Colour Change Negative Result
#6 – Lactose Solution Dark Yellow Colour Change Negative Result
#7 – 1% Glycogen Solution Dark Orange-Brown Colour Change Positive Result
#8 – 1% Starch Solution Violet-Blue Colour Change Positive Result
#9 – Protein Dark Yellow Colour Change Negative Result
#10 – Beer Dark Yellow Colour Change Negative Result
#11 – Distilled Water Dark Yellow Colour Change Negative Result
#12 – Unknown Solution: #10 Dark Yellow Colour Change Negative Result

Table 1.2: Benedict’s Test Results
Benedict’s Test was preformed to find the presence of reducing sugars (contain a free aldehyde or ketone group) in the 11 known sample solutions and the one unknown sample solution. The positive result for the existence of a reducing sugar in the samples was the colour change of orange-red-brown plus precipitate.

SAMPLE SOLUTIONS COLOUR CHANGE RESULTS POSITIVE/NEGATIVE
#1- 1% Glucose Solution Red-Orange-Brown Colour Change + Precipitate Positive Result
#2 – 0.3% Glucose-1-Phosphate Light Blue Colour Change Negative Result
#3 – 1% Maltose Solution Red-Orange-Brown Colour Change + Precipitate Positive Result
#4 – Honey Solution Dark Brown Colour Change + Precipitate Positive Result
#5 – Sucrose Solution Light Blue Colour Change Negative Result
#6 – Lactose Solution Red-Orange-Brown Colour Change + Precipitate Positive Result
#7 – 1% Glycogen Solution Light Blue Colour Change Negative Result
#8 – 1% Starch Solution Light Blue Colour Change Negative Result
#9 – Protein Dark Blue Colour Change Negative Result
#10 – Beer Light Brown Colour Change + Precipitate Positive Result
#11 – Distilled Water Light Blue Colour Change Negative Result
#12 – Unknown Solution: #10 Red-Orange-Brown Colour Change + Precipitate Positive Result

Table 1.3: Biuret Test Results
The Biuret Test was preformed to find the presence of proteins in the 11 known sample solutions and the one unknown sample solution. The positive result for the existence of a protein in the samples was the colour change of violet-blue.

SAMPLE SOLUTIONS COLOUR CHANGE RESULTS POSITIVE/NEGATIVE
#1- 1% Glucose Solution Light Blue Colour Change Negative Result
#2 – 0.3% Glucose-1-Phosphate Light Blue Colour Change Negative Result
#3 – 1% Maltose Solution Light Blue Colour Change Negative Result
#4 – Honey Solution Light Green/Yellow Colour Change Negative Result
#5 – Sucrose Solution Light Blue Colour Change Negative Result
#6 – Lactose Solution Light Blue Colour Change Negative Result
#7 – 1% Glycogen Solution Light Blue Colour Change Negative Result
#8 – 1% Starch Solution Light Blue Colour Change Negative Result
#9 – Protein Violet-Blue Colour Change Positive Result
#10 – Beer Brown-Green-Yellow Colour Change Negative Result
#11 – Distilled Water Light Blue Colour Change Negative Result
#12 – Unknown Solution: #10 Light Blue Colour Change Positive Result

DISCUSSION After testing and recording the results of all three tests the following results have been concluded. Solution sample number one, 1% glucose solution, tested negative for the Iodine Test, the presence of starch and glycogen; and the Biuret Test, the presence of proteins.
The 1% glucose solution did indeed test positive for Benedict’s Test of reducing sugars. Glucose being a monosaccharide has a free reactive carbonyl group allowing it to react with the copper sulfate in the Benedict’s solution. (Gurien, 2008) The same results and reasoning can be applied to sample solution number three: 1% maltose solution; sample solution number four: honey; sample solution number six: lactose solution, and the unknown solution sample [#10]. The entire sample solutions named above contains some form of monosaccharide or disaccharide, which helps prove the theory that they are reducing sugars.
Based upon the results of the unknown sample solution, it can be concluded that it is indeed a reducing sugar since it tested positive for Benedict’s test. It can either be glucose, maltose or lactose. The unknown sample solution cannot be identified as honey since it the unknown solution was colourless and honey was not colourless.
Sample solution number two, 0.3% glucose-1-phosphate, tested negative for the Iodine Test, the presence of starch and glycogen; for Benedict’s Test, the presence of reducing sugars; and for the Biuret Test, for proteins. Sample solution number two did not test positive for any of the macromolecule tests that were executed. The conclusion for this sample solution is that because it contains a phosphate molecule, it is considered a part of a nucleic acid. None of the three tests were a test for nucleic acids therefore it is correct to believe that it is apart of the nucleic acid macromolecule.
Sample solution number five, sucrose solution, tested negative for the Iodine Test, presence of starch and glycogen; Benedicts Test, presence of reducing sugars; and the Biuret Test, for the presence of proteins. Sucrose is indeed a sugar and it would seem as though it would test positive for Benedict’s Test. It can be concluded that even though sucrose is a disaccharide; it does not have a free, unoccupied aldehyde of ketone group. (Clark, 2004) (Gurien, 2008)
Glycogen, sample solution number seven, did accurately test positive for the Iodine Test, and negatively for both Benedict’s Test and the Biuret Test. Sample solution number eight, starch, also ended up with the same results as glycogen. They both are exactly what the Iodine’s Test is intended to test for. Starch is a polysaccharide found in plants while glycogen is a polysaccharide found in animals. They both contain glycosidic linkages, which helps them react with the iodine solution. (Gurien, 2008) (Lea, 2002)
Sample solution number nine: protein, did accurately test positive for the Biuret Test , and negatively for both Benedict’s Test and the Iodine Test. Proteins contain a peptide bond which positively reacts with the biuret compound. (Gurien, 2008) (Lea, 2002)
Sample solution number ten: beer, tested negative for both the Iodine Test and the Biuret Test; but positive for Benedict’s Test. Beer should have positively reacted with the Iodine Test because it does in fact contain yeast, which is considered a starch. This result may have been affected by a cause of error. The sample solution could have been mistakenly mixed with another solution that it should have not been mixed with. (Gurien, 2008)
Sample solution number eleven: water, was the negative controlling substance for all of the tests. Distilled water contained no forms of macromolecules that would react with any of the three tests. All of the results were based upon water being the negative controlling substance.
In conclusion most of the sample solutions reacted correctly to each of the tests with an exception to sample solution number 10: beer. The Iodine Test did in fact test positive for glycogen and starch, sample solutions seven and eight. Benedict’s Test did correctly test positive for reducing sugars, glucose, maltose, honey and lactose. Lastly, the Biuret Test did test positive for the protein sample solution.

REFERENCES Clark, Jim. (2004). “Oxidation of Aledehydes and Ketones” 29 September 2010.

Davidson, John K. (2000). Clinical diabetes mellitus: a problem-oriented approach. 3rd Ed. Theieme Medical Publishers, Inc. New York. pp. 250.

Department of Biology 2001 Introductory Cell Biology Laboratory Manual. University of Waterloo, Waterloo. pp. 17-20.

Gardner, A.L. (1997). “Gerty Cori, Biochemist, 1896-1957,” in Matyas, M.L. & Haley-Oliphant, A.E. (Editors). (1997). Women Life Scientists: Past, Present, and Future – Connceting Role Models to the Classroom Curriculum. Bethseda, MD: American Physiological Society, p.291-306

Gurien, M. (09 August 2008) “Biuret Test” Ohio University. 29 September 2010.

Karp, G. E. (2008). Cell and Molecular Biology. 5th Ed. John Wiley & Sons, Inc. New Jersey. pp. 40-50; 74-75.

Lea, C. (2002) “Biological Macromolecules” University of New Mexico. 29 September 2010.

Ophardt. C.E. (2003) “Starch-Iodine” Elmhurst College. 29 September 2010.

References: Clark, Jim. (2004). “Oxidation of Aledehydes and Ketones” 29 September 2010. Davidson, John K. (2000). Clinical diabetes mellitus: a problem-oriented approach. 3rd Ed. Theieme Medical Publishers, Inc. New York. pp. 250. Department of Biology 2001 Introductory Cell Biology Laboratory Manual. University of Waterloo, Waterloo. pp. 17-20. Gardner, A.L. (1997). “Gerty Cori, Biochemist, 1896-1957,” in Matyas, M.L. & Haley-Oliphant, A.E. (Editors). (1997). Women Life Scientists: Past, Present, and Future – Connceting Role Models to the Classroom Curriculum. Bethseda, MD: American Physiological Society, p.291-306 Gurien, M. (09 August 2008) “Biuret Test” Ohio University. 29 September 2010. Karp, G. E. (2008). Cell and Molecular Biology. 5th Ed. John Wiley & Sons, Inc. New Jersey. pp. 40-50; 74-75. Lea, C. (2002) “Biological Macromolecules” University of New Mexico. 29 September 2010. Ophardt. C.E. (2003) “Starch-Iodine” Elmhurst College. 29 September 2010.