Proteins Maria Virginia M. Orticio Biology Department October 3, 2012
Abstract 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 re actions 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, 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(NH 2)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
added with 3 ml 10% NaOH solution and a very dilute CuSO 4 drop by drop while shaking for every drop. In Millon’s test, 5 drops of Millon’s reagent was added and heat was applied to the mixture. For Xanthoproteic test, 3 drops of concentrated solution was added and heat was applied to the mixture. After heating, the mixture was then cooled and was made slightly basic by adding NH4OH solution. In sulfur test, 10 drops of 10% NaOH solution and 1 ml of saturated lead acetate solution was added. After adding the solutions, it was placed in a boiling water bath. Under precipitation reactions, another set of subtests should be conducted: by heat, with strong mineral acids and with salt of a heavy metal. Again, these tests require a 3 ml egg albumin solution. For the first test, heat was applied for a few minutes then observe. For the test with strong mineral acids, 5 ml of concentrated HNO 3 was added slowly along the tube while in an inclined position. In the test with salt of heavy metals, 1% HgCl 2 solution was added drop by drop while shaking until visible change can be observed. Results and Discussion Tests
Results/Observations
A.
Burnt meat-like smell after heating the egg albumin powder. Turns the color of the powder into brownish black. White gelatinous homogenous mixture was formed after mixing the egg albumin with water.
B.
C.
BURN TEST
EGG ALBUMIN SAMPLE
COLOR REACTION
1.
Biuret Test
Blue-Violet coloration
2.
Millon’s Test
Formation of precipitate
3.
Xanthoproteic Test
Intense yellow coloration after addition of NH 4OH
cloudy
white
4. D.
Sulfur Test
Black Precipitate
PRECIPITATION REACTION
1.
By Heat
Formation of white solid
2.
With Strong Mineral Acids
Yellow junction
3.
With Salt of a Heavy Metal
Colorless white precipitate
bubbles
and
Burning Test For the burning test of proteins, the egg albumin powder was heated and the color of the powder turns into brownish black. The reason for this coloration is due to the presence of amino acids, glycine and lysine, on the egg albumen. Glycine is an amino acid that is when reacted with sugar can produce a brown coloration, this is the Maillard reaction. The coloration is made up of melanoidins which is a large molecule that is polymerized from the products of the Maillard reaction. Lysine is also an amino acid that produces most of the brown coloration when reacting with sugars, specifically five carbon sugars or ribose. Maillard reaction is also responsible for the aroma of burnt meat in the heating of egg albumin powder. Preparation of Egg Albumin Sample As for the preparation of egg albumin sample, a mixture of egg white and water was mixed and later then filtrated. The formation of gelatinous substance on this solution is due to the reason that egg albumin protein contains amino acids, and these amino acids on egg albumin can be both hydrophobic and hydrophilic amino acids. When these amino acids are mixed with water, hydrophobic amino acids are being packed together at the center away
from the water while hydrophilic amino acids are on the side closer to the water. Color reactions of Proteins Proteins contain peptide bonds and amino acid residues. Due to this, proteins react with variety of reagents to form color reactions. 1. Biuret Test- This test confirms the presence of peptide bond and will result to a violet coloration of the solution. This is a general test for identification of proteins. In alkaline medium, CuSO4 reacts to form a violet colored complex. This test is so named since this reaction is given by the substance biuret, which is obtained by condensation of 2 molecules of urea when heated in 180 degrees Celsius. 2. Millon’s Test- Millon’s reagent is made up by dissolving Hg in HNO 3 and diluting with water. Other source shows a different chemical solution for Millon’s reagent: Mercuric sulfate in Sulfuric Acid (http://amrita.vlab.co.in/?sub=3&brc h=63&sim=1094&cnt=1 ). But both reagents confirm the presence of the hydroxyphenyl group of tyrosine, the only amino acid that contains a phenol group on which a hydroxyl group is attached. It both gives a red precipitate. 3. Xanthoproteic Test- This test confirms the presence of a phenyl ring in amino acids, specifically the phenylalanine, tyrosine and tryptophan groups of amino acids. These amino acids reacts with concentrated HNO 3 at high temperature to form nitrocompounds which are yellow in color, it turns
into intense yellow-orange color in alkaline medium. 4. Sulfur Test- This test confirms the presence of sulfur-containing proteins, cysteine and cystine, which gives a black or brown precipitate positive result. When these proteins are boiled with strong alkali, organic sulfur is converted into sodium sulfide. Sodium sulfide will react with lead acetate to form lead sulfide precipitate (insoluble), which is the one responsible for its positive result of black/brown precipitate. Precipitation reactions Proteins have either a positive or a negative charge depending on the solution. The stability of the proteins in the solution will depend mainly on the charge and hydration. Proteins form a colloidal solution in which a cloud of water molecule surrounds the protein molecule. 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. Proteins can be precipitated by dehydration or neutralizing of the charges. 1. By heat- When you apply heat, you agitate those placidly drifting eggwhite proteins, bouncing them around. They slam into the surrounding water molecules. All this bashing about breaks the weak bonds that kept the protein curled up. The egg proteins uncurl and bump into other proteins that have also uncurled. New chemical bonds form but rather than binding the protein to itself, these bonds connect one protein to another. After enough of this bashing and bonding, the solitary
egg proteins are solitary no longer. They’ve formed a network of interconnected proteins. The water in which the proteins once floated is captured and held in the protein web. If you leave the eggs at a high temperature too long, too many bonds form and the egg white becomes rubbery. 2. With Strong Mineral Acids- This forms a white ring between the 2 junctions. And albumin is precipitated by nitric acid. Nitric acid causes denaturation of proteins with the formation of a white precipitate (this differs from the nitration reaction in “xanthoproteic acid test”). 3. With Salts of a Heavy Metal- Heavy 2+ 2+ 2+ metals (e.g. Hg , Pb , Cu ) are high molecular weight cations. The positive charge of these cations counteracts the negative charge of the carboxylate group in proteins giving a precipitate.
As mentioned earlier to the conclusions, 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. The students should always observe the proper and correct procedure of the experiments conducted for them to obtain a positive and expected result. References
Conclusions and Recommendations
The results obtained from all of the experiments shows that the above tests confirm the presence of proteins. The result matches the expected outcomes of every test and was observed properly. There is just one experiment that presented a different result, the Millon’s test. 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 HNO3. Instead, our result produced a brown coloration. The only possible reason for this incident is that, the solution was exposed too much to heat and all the proteins molecules collapsed. 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.
http://kitchenscience.scitoys.com/heating http://thescienceinformant.wordpress. com/2010/12/13/moleculargastronomy-food-colours-andengineered-fare/ http://www.exploratorium.edu/cooking /eggs/eggscience.html http://books.google.com.ph/books?id= nPphGdHO_XcC&pg=PA15&source=gbs _toc_r&cad=4#v=onepage&q&f=false http://faculty.ksu.edu.sa/75115/BCH%2 0221Lectures/Enzyme%20lectures%20f or%20Cat%201.pdf http://www.books-aboutcalifornia.com/Pages/Experimental_Org anic_Chemistry/Ex_Organic_Chem_Cha p_26.html http://www.pua.edu.eg/Version2/Cour ses2/Dentistry%20Courses/Freshmen/S pring/BCM101/Practical/Week%204%2 0practical%20_Chemistry%20of%20prot eins_.pdf http://en.wikipedia.org/wiki/Protein http://amrita.vlab.co.in/?sub=3&brch= 63&sim=1094&cnt=1
http://www.wisegeek.com/what-isalbumin.htm
