Experiment No.

2 AMINO ACIDS AND PROTEINS (Part I)

Monica Francine Jose 08-8031 Ana Marie Rosqueta 08-8293 Zarah Krizcia Ruiz 08-821

January 7, 2009 Chemistry 42: Biochemistry

ABSTRACT Proteins are macromolecules that are made up of amino acids that are linked together by peptide bonds. In this experiment we studied some basic properties of amino acids and proteins. We isolated the protein from natural sources. We also conducted solubility test ans color reactions of intact proteins. Proteins do not contain some amino acis that can resulty to a positive result in some tests. The first part of the experiment was conducted 7th day of January 2009.

INTRODUCTION Objective and Purpose 1. To be able to know some of the different properties of proteins 2. To determine the solubility of some amino acids and proteins. 3. To determine the color reactions of intact proteins. Theory Proteins are organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids, however in certain organisms the genetic code can include selenocysteine - and in certain archaea - pyrrolysine. The residues in a protein are often observed to be chemically modified by post-translational modification, which can happen either before the protein is used in the cell, or as part of control mechanisms. Proteins can also work together to achieve a particular function, and they often associate to form stable complexes. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in animals' diets, since animals cannot synthesize all the amino acids they need and must obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism. In Biochemistry, proteins are linear polymers built from 20 different L--amino acids. All amino acids possess common structural features, including an carbon to which an amino group, a carboxyl group, and a variable side chain are bonded. Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CONH amide moiety into a fixed conformation. The side chains of the standard amino acids, detailed in the list of standard amino acids, have different chemical properties that produce threedimensional protein structure and different reactivities, are therefore critical to protein function.

The amino acids in a polypeptide chain are linked by peptide bonds. Once linked in the protein chain, an individual amino acid is called a residue, and the linked series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone. The peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are roughly coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone. Due to the chemical structure of the individual amino acids, the protein chain has directionality. The end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus, whereas the end with a free amino group is known as the N-terminus or amino terminus. The words protein, polypeptide, and peptide are a little ambiguous and can overlap in meaning. Protein is generally used to refer to the complete biological molecule in a stable conformation, whereas peptide is generally reserved for a short amino acid oligomers often lacking a stable three-dimensional structure. However, the boundary between the two is not well defined and usually lies near 2030 residues. Polypeptide can refer to any single linear chain of amino acids, usually regardless of length, but often implies an absence of a defined conformation. An amino acid is a molecule containing both amine and carboxyl functional groups. These molecules are particularly important in biochemistry, where this term refers to alphaamino acids with the general formula H2NCHRCOOH, where R is an organic substituent. In the alpha amino acids, the amino and carboxylate groups are attached to the same carbon, which is called the carbon. The various alpha amino acids differ in which side chain (R group) is attached to their alpha carbon. They can vary in size from just a hydrogen atom in glycine through a methyl group in alanine to a large heterocyclic group in tryptophan. Amino acids are critical to life, and have a variety of roles in metabolism. One particularly important function is as the building blocks of proteins, which are linear chains of amino acids. Amino acids are also important in many other biological molecules, such as forming parts of coenzymes, as in S-adenosylmethionine, or as precursors for the biosynthesis of molecules such as heme. Due to this central role in biochemistry, amino acids are very important in nutrition. Some test are use to determine the color reactions of intact proteins. These include:

Ninhydrin Test: Ninhydrin is a powerful oxidizing agent reacts with amino acids, between pH 48 to give a purple color complex. Ninhydrin reagent is reduced to hydrindantin during reaction with amino acids. The amino acid in turn is converted into an aldehyde. Ammonia Carbon dioxide is evolved. Hydrindantin and ammonia interact with another molecule of ninhydrin to form Ruhemanns purple colored complex. Millons Test: Phenolic amino acid on treatment with Millons reagent gives red color. Mercuric sulfate forms a colored compound with hydroxyl group of amino acid (Tyrosine). Hopkins Cole Test: This reaction is answered by tryptophan. This reaction is due to the presence of indole group in tryptophan. Sulfur Test: Amino acids containing the thiol or sulphydryl group reacts with sodium plumbate to form a dark grey or black precipitate which is insoluble in dil.HCl. Biuret Test: The biuret test is a chemical test used for detecting the presence of peptide bonds. In a positive test, a copper (II) ion is reduced to copper (I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. It is possible to use the Biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.

EXPERIMENTAL (Materials and Methods) I. Isolation of Proteins 1. Isolation of Gluten from Wheat Flour: a. Add water in small quantities to 50g of wheat flour until thick, dough is obtained. b. Knead thoroughly with the hand and soak it in water for about 30 seconds. Take it out from the water and continue to knead it in your hands. This process is done to remove by washing, the starch from the flour. Take note of the color of the water that is dripping from your hands, it indicates the starch is being removed. Repeat the process until the starch is removed. c. Take a small portion of gluten, place it in a test tube and add 2ml of concentrated HCl. Heat the mixture until the gluten is dissolved and then neutralize it with 2ml of concentrated NaOH. Refrigerate and reserve the solution for the color reactions of proteins. 2. Isolation of Bean Protein: a. Sort and wash cup of beans. b. Soak in water overnight. c. Grind the beans finely in a mortar and then blend with water to make a thick paste. d. Squeeze the milk through a cotton cloth and discard the insoluble residue. e. Add 10ml of 1N acetic acid until precipitation is complete. Let the mixture stand undisturbed until the precipitate settles to the bottom. f. Strain through a thick cotton cloth and squeeze out as much water as possible. g. Dry the precipitate. 3. Precipitation of Powdered Egg Albumin: a. Separate the white of the egg and finely divide it with an egg beater. b. Treat with 20ml of water. Let it stand to allow the precipitate to sttle and filter. c. Evaporate the filtrate to dryness on a water bath at 50C. d. Powder the residue in a mortar. II. Solubility 1. Test separately the solubility of approximately 0.1g of powdered egg albumin, bean protein and casein in 1ml of the following solvents: water, saturated NaCl, 0.1N HCl, and 0.1N NaOH. 2. Place about 0.1g of the powdered egg albumin in a dry test tube and heat in a boiling water bath for 5 minutes. Let it cool and test its solubility with each of the solvent above.

3. Repeat procedure #2 but add 1ml of water to the egg albumin before heating in the water. III. Color Reactions of Intact Proteins 1. Ninhydrin Reaction: a. To 10 drops of the sample in a 100ml beaker add 0.1M NaC2H3O2 buffered at pH 5.0. Add 1-2 drops of 0.1% prepared ninhydrin solution. b. Cover with a watch glass and heat to boiling in a water bath for 1-2 minutes. c. Allow to cool and observe the results. 2. Biuret Test: a. Add 1ml of 2.5N NaOH to 3ml of the protein suspension and mix. b. Add a drop of 0.01M CuSO4 and observe the result. 3. Xantoproteic Test or Yellow Test: a. To 3ml of the protein suspension, add slowly 1ml of concentrated HNO3. b. Heat gently. Cool the solution with flowing water and add slowly, drop by drop concentrated NaOH. Continue the addition and observe carefully the changes involved. Stop adding the alkali if the solution is already alkaline. 4. Millons Test: a. Add 5 drops of fresh Millons reagent to 3ml of the protein suspension. b. Carefully heat the mixture and observe he different changes involved. 5. Hopkins-Cole Test: a. Mix 2ml of the reagent to 3ml of the protein suspension in a test tube. b. Incline the tube and add slowly 5ml of concentrated H2SO4, letting it flow down the inside of the tube so that two layers will form. 6. Sulfur Test: a. Prepare 6 test tubes and add the following solution using pipette.

Solution (ml) Distilled water 1% glycine solution 1% cysteine 1% methionine 1%casein Diluted egg white solution in 0.85% saline (1:10) Distilled water 40% 2.5 1 drop 2.5 1 drop 1 0.5 2 0.5 -

Test Tube No. 3 4 0.5 0.5 -

5 0.5 -

6 -

0.5

2.5 1 drop

2.5 1 drop

2.5 1 drop

2.5 1 drop

NaOH b. Mix well and add 2 drops of 10% lead acetate in every test tube. c. Mix well, and then boli in water bath at 100C for 5 minutes. d. Observe and report the results (compare with test tube 1). Test tube 1 is the control.

RESULTS AND DISCUSSION Table 1. Solubility of some proteins

Nonheating Water Saturated NaCl 0.1N HCl 0.1N NaOH insoluble insoluble insoluble insoluble

Egg albumin Dry heating insoluble insoluble insoluble insoluble

Heating with water insoluble insoluble insoluble insoluble

Bean protein

Casein insoluble insoluble insoluble insoluble

As a general rule, proteins that are undenatured are most soluble. Heating proteins results in protein denaturation, decreasing solubility. Generally, as the water content of a solution decreases, the amount of heat required to denature protein increase. The protein is insoluble in all solvents but the degree. The solubility of the protein decreases when it is heated making it insoluble. Table 2. Confirmatory tests for proteins Egg albumin Positive Positive Positive Positive Positive Observations and results Bean protein Casein Positive Positive Positive Positive Positive

Ninhydrin reaction Biuret test Millons test Hopkins-cole test Sulfur test

In Ninhydrin test, the resulting color is blue violet. This indicates a positive reaction. Hydrindantin and ammonia interact with another molecule of ninhydrin to form Ruhemanns purple colored complex. It also indicates the presence of amino acid. In Biuret test, the resulting color is dark purple. This indicates a positive reaction. In a positive test, a copper (II) ion is reduced to copper (I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. In Millons test, the resulting color is red. This indicates a positive reaction. Presence of phenolic group containing amino acid (tyrosine) in the protein In Hopkins-cole test the resulting color is yellow. This indicates a positive reaction. This is due to the presence of tryptophan.

In Sulfur test, a black precipitate is formed. This indicates the presence of Cysteine in the protein. Post Laboratory Guide Questions: 1. What type of chemical grouping is present in all proteins?
a. Amines. All proteins contain hydrogen, carbon, oxygen, and more importantly

nitrogen. Proteins being made from amino acids contain the amino group and the carboxyl group.
2. How many of these chemical groupings must be present in a molecule to give Biuret

test? a. Biuret test is used for detecting the presence of peptide bonds. So, two chemical groupings must be present. Biuret reagent is made of sodium hydroxide and copper sulfate. It is blue in color but it turns violet in the presence of proteins and changes to pink when combines with short-chainpolypeptides.

3. Give the principle involved and the chemical structure responsible for a positive Biuret test.
a. Biuret test is used for detecting the presence of peptide bonds. It relies on the

reduction of copper(II) ions to copper(I), the latter form a complex with the nitrogen of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins.
4. Do all amino acids having an aromatic ring give a positive Xantoproteic test under

identical conditions? Give reasons to your answer. a. Yes. Because these rings undergo nitration when treated with the Xanthoproteic test. 5. Do most proteins give a positive Xanthoproteic test? a. Yes. Beacuse nearly all proteins contain aromatic rings.

6. What chemical structure is necessary for a positive Millons test? For a HopkinsCole test?
a. For Millons test: Phenol. b. For Hopkins-Cole test: indole group of tryptophan. 7. What is the role of concentrated H2SO4 in the Hopkins-Cole test?

a. It hydrolyzes the protein solution at the interface. The indole ring reacts with glyoxylic acid in the presence of a strong acid to foem a violet cyclic product. TheHopkins-Cole reagent only reacts with proteins containing tryptophan. The protein solution is hydrolyzed by the concentrated sulphuric acid at the solution interface. Once the tryptohan is free, it reacts with the glyoxylic acid to form violet product.

REFERENCES A. Internet
a. http://www.chemistryexplained.com/Pr-Ro/Protein-Solubility.html b. http://www.innovatewithdairy.com/InnovateWithDairy/Articles/FAQ_ProteinSol

ub_032905.htm
c. http://www.chemistryexplained.com/Pr-Ro/Proteins.html d. http://en.wikipedia.org/wiki/Amino_acid e. http://en.wikipedia.org/wiki/Protein f. http://www.cem.msu.edu/~reusch/VirtualText/proteins.htm g. http://www.chemistry.mcmaster.ca/~chem2o6/labmanual/expt11/2o6exp11.html h. http://www.cerlabs.com/experiments/10875404480.pdf i. j.

http://en.wikipedia.org/wiki/Biuret_test http://faculty.ksu.edu.sa/75115/BCH%20221Lectures/quaalitative%20test%20for %20protein.pdf