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Maria Virginia M. Orticio Biology Department October 3, 2012

Proteins are essential to every organism since these organic molecules facilitate almost all of the cellular activities within every living cell. Proteins are made up of long chains of amino acids which contain both an amino and carboxylic groups and are both considered “acid” and “base” making it an amphoteric. These amino acids are linked together by peptide bonds. And these peptide bonds are responsible for the color reactions of proteins. Giving the biuret, millon’s, xanthoproteic, and sulfur tests a different color confirmatory result each. Another test for the detection of proteins is the precipitation reaction. These tests confirm the presence of proteins through precipitation due to the reaction of negatively charged protein in the different charges of the reagents used. The results obtained match the expected outcome for every test except for the millon’s test which produces a brown coloration, instead of a reddish-brown or brick red, due to prolonged heating.

Introduction Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form facilitating a biological function. Proteins are made up of amino acids and these organic compounds contain both an amino and a carboxyl groups making it both base and acid calling it amphoteric. It has the general formula of RCH(NH2)COOH. These proteins are essential to every organism since most of the cellular activities are facilitated by proteins. This experiment aims to determine the different properties and reactions of proteins and amino acids, the reagents responsible for these reactions. The following tests to be performed should have a positive result since these tests are specifically done to detect the presence of proteins and amino acids. Peptide bonds of the amino acids are the one responsible for the color reactions of proteins while the

negative charge of the protein is responsible for its precipitation reaction. Methodology There are different tests that were conducted to determine the different properties of proteins such as; (a) burning test, (b) color reactions, (c) precipitation reactions. For (a) burning tests, a small amount of egg albumin powder was placed in an evaporated dish and heat was applied. Then, the odor was observed. In order to proceed to (b) color reactions and (c) precipitation tests, I prepared an egg albumin sample by separating the white component from the yolk component; 150-ml distilled water was added and mixed thoroughly. Then, the mixture was filtered and the filtrate can now be used for the following procedures. Color reactions consist of several tests: Biuret, Millon, Xanthoproteic and Sulfur test. For these following test 3 ml egg albumin solution was used. In Biuret test, it was

a mixture of egg white and water was mixed and later then filtrated. Sulfur Test PRECIPITATION REACTION By Heat With Strong Mineral Acids With Salt of a Heavy Metal Black Precipitate 1. Maillard reaction is also responsible for the aroma of burnt meat in the heating of egg albumin powder. D. The reason for this coloration is due to the presence of amino acids. heat was applied for a few minutes then observe. it was placed in a boiling water bath. In Millon’s test. 3 drops of concentrated solution was added and heat was applied to the mixture. In sulfur test. Preparation of Egg Albumin Sample As for the preparation of egg albumin sample. and these amino acids on egg albumin can be both hydrophobic and hydrophilic amino acids. After heating. When these amino acids are mixed with water. Turns the color of the powder into brownish black. 5 drops of Millon’s reagent was added and heat was applied to the mixture. 5 ml of concentrated HNO3 was added slowly along the tube while in an inclined position. 2. BURN TEST 4. another set of subtests should be conducted: by heat. For the first test. After adding the solutions. the egg albumin powder was heated and the color of the powder turns into brownish black. Results and Discussion Tests A. EGG ALBUMIN SAMPLE C. 10 drops of 10% NaOH solution and 1 ml of saturated lead acetate solution was added. Lysine is also an amino acid that produces most of the brown coloration when reacting with sugars.added with 3 ml 10% NaOH solution and a very dilute CuSO4 drop by drop while shaking for every drop. glycine and lysine. Under precipitation reactions. White gelatinous homogenous mixture was formed after mixing the egg albumin with water. 3. on the egg albumen. specifically five carbon sugars or ribose. COLOR REACTION Biuret Test Millon’s Test Xanthoproteic Test Blue-Violet coloration Formation of cloudy white precipitate Intense yellow coloration after addition of NH4OH 1. with strong mineral acids and with salt of a heavy metal. 2. hydrophobic amino acids are being packed together at the center away Results/Observations Burnt meat-like smell after heating the egg albumin powder. Formation of white solid Yellow junction bubbles and 3. For Xanthoproteic test. Glycine is an amino acid that is when reacted with sugar can produce a brown coloration. the mixture was then cooled and was made slightly basic by adding NH4OH solution. The formation of gelatinous substance on this solution is due to the reason that egg albumin protein contains amino acids. these tests require a 3 ml egg albumin solution. Colorless white precipitate Burning Test For the burning test of proteins. For the test with strong mineral acids. The coloration is made up of melanoidins which is a large molecule that is polymerized from the products of the Maillard reaction. 1% HgCl2 solution was added drop by drop while shaking until visible change can be observed. . In the test with salt of heavy metals. B. this is the Maillard reaction. Again.

you agitate those placidly drifting eggwhite proteins. This is a general test for identification of proteins. These amino acids reacts with concentrated HNO3 at high temperature to form nitrocompounds which are yellow in color. 1.vlab. 3. In alkaline medium. the solitary .Millon’s reagent is made up by dissolving Hg in HNO3 and diluting with water. 2. proteins react with variety of reagents to form color reactions. Polar groups of the protein tend to attract the polar groups of the water molecule around them by hydrogen bond to produce a shell of hydration. cysteine and cystine. After enough of this bashing and bonding. This test is so named since this reaction is given by the substance biuret. it turns into intense yellow-orange color in alkaline h=63&sim=1094&cnt=1). which is obtained by condensation of 2 molecules of urea when heated in 180 degrees Celsius. Due to this. which gives a black or brown precipitate positive result. 4. The egg proteins uncurl and bump into other proteins that have also uncurled. CuSO4 reacts to form a violet colored complex.This test confirms the presence of sulfur-containing proteins.This test confirms the presence of a phenyl ring in amino acids.When you apply heat. The stability of the proteins in the solution will depend mainly on the charge and hydration. Sodium sulfide will react with lead acetate to form lead sulfide precipitate (insoluble). the only amino acid that contains a phenol group on which a hydroxyl group is attached. Precipitation reactions Proteins have either a positive or a negative charge depending on the solution. bouncing them around. tyrosine and tryptophan groups of amino acids. Millon’s Test. They slam into the surrounding water molecules. Xanthoproteic Test. By heat. Proteins can be precipitated by dehydration or neutralizing of the charges. which is the one responsible for its positive result of black/brown precipitate. New chemical bonds form but rather than binding the protein to itself. All this bashing about breaks the weak bonds that kept the protein curled up.from the water while hydrophilic amino acids are on the side closer to the water. 1. Sulfur Test. these bonds connect one protein to another. Biuret Test. organic sulfur is converted into sodium sulfide.This test confirms the presence of peptide bond and will result to a violet coloration of the Color reactions of Proteins Proteins contain peptide bonds and amino acid residues. Other source shows a different chemical solution for Millon’s reagent: Mercuric sulfate in Sulfuric Acid (http://amrita. It both gives a red precipitate. But both reagents confirm the presence of the hydroxyphenyl group of tyrosine. specifically the phenylalanine. When these proteins are boiled with strong alkali. Proteins form a colloidal solution in which a cloud of water molecule surrounds the protein molecule.

com/2010/12/13/moleculargastronomy-food-colours-andengineered-fare/ http://www. the Millon’s test.pdf 0221Lectures/Enzyme%20lectures%20f or%20Cat%201.This forms a white ring between the 2 The only possible reason for this incident is that. The water in which the proteins once floated is captured and held in the protein web. The result matches the expected outcomes of every test and was observed properly. As mentioned earlier to the conclusions.Heavy metals (e.html http://books. If you leave the eggs at a high temperature too anic_Chemistry/Ex_Organic_Chem_Cha /eggs/eggscience. The positive charge of these cations counteracts the negative charge of the carboxylate group in proteins giving a Hg2+. Nitric acid causes denaturation of proteins with the formation of a white precipitate (this differs from the nitration reaction in “xanthoproteic acid test”) ses2/Dentistry%20Courses/Freshmen/S pring/BCM101/Practical/Week%204%2 0practical%20_Chemistry%20of%20prot http://thescienceinformant. The expected result should produce a reddish brown or brick red coloration on the solution due to the reaction of hydroxyphenyl group with the Hg in 63&sim=1094&cnt=1        . Instead. References   http://kitchenscience.exploratorium.pdf http://en. too many bonds form and the egg white becomes rubbery.scitoys. This should be noted and should be included to the recommendation as I have suggested and as for all the reagents to be used for other experiments.g. our result produced a brown coloration. With Salts of a Heavy Metal. The students should always observe the proper and correct procedure of the experiments conducted for them to obtain a positive and expected result. There is just one experiment that presented a different result. Cu2+) are high molecular weight nPphGdHO_XcC&pg=PA15&source=gbs _toc_r&cad=4#v=onepage&q&f=false http://faculty. They’ve formed a network of interconnected proteins. And albumin is precipitated by nitric acid. the only recommendation I should include to this report is to avoid prolonged exposure to heat of the solutions or reagents. This may cause a different result leading to an unsuccessful experiment.wikipedia. Conclusions and Recommendations The results obtained from all of the experiments shows that the above tests confirm the presence of proteins.egg proteins are solitary no longer. 3.html http://www. the solution was exposed too much to heat and all the proteins molecules collapsed. With Strong Mineral

wisegeek. http://www.htm .com/what-isalbumin.