Characterization of Intact Protein and Base Hydolyzate

through color reaction tests Ma. Bianca Therese Relova, Doxa Dave Rotap*, Bea Rosalinni Santiago, and Ramon Arvin Noriel Santos Chemistry Department, College of Science, Main Building, University of Santo Tomas, Espaa Manila, Philippines ABSTRACT Because amino acids are essential for every living being, to know what kind of amino acids are present in a product are essential. Through color reaction tests, an excellent source of proteins was tested for kinds of amino acids present.

INTRODUCTION Amino acids are the building blocks of Proteins, macromolecule essential for every living being. These Proteins (which are actually chains of amino acids) are like Legos for our life. There are a total of 20 different kinds of amino acids that form these proteins. The kinds of amino acids determine the shape and the function of the proteins formed. As there are different amino acids, there are also different chains of amino acids and these chains contribute to the ability of the protein formed. Our body has the ability to produce most of the amino acids that we need. These amino acids are known as Nonessential Amino Acids. These monomers include Alanine, Arginine, Asparagine, Aspartate, Cysteine, Glutamate, Glutamine, Glycine, Proline, Serine, and Tyrosine. All of these are ably synthesizes by our own body. Some amino

acids cannot be synthesized by our body. These are the so-called Essential Amino Acids. As the name implies, our body needs these amino acids in order to function and grow properly. These include Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine. These amino acids can only be supplied through ingestion of food. Milk is an excellent source of casein. Casein exists as a multisubunit protein complex dispersed throughout the fluid phase of milk. To determine if proteins are truly present and what kinds of amino acids are present in the casein, a series of tests are conducted. The test would have the precipitated intact casein and an autoclaved base hydrolyzate taken from the same casein as the samples. An autoclaved casein will undergo Maillard reaction where in the amino group of an amino acid reacts with a carbonyl group of a sugar, fusing the two molecules together. In effect, the amino acids present in the autoclaved casein are disrupted and altered. This would lessen the amino acids. The autoclaved casein was base hydrolyzed, releasing ions and making the solution neutral in pH. It was also filtered so only the liquid portion of the base hydrolyzate was used in the experiment.

RESULTS AND DISCUSSION Color reaction tests Intact Casein Biuret test Violet Solution Base Hydrolyzate Blue Solution

Sakaguchi test

Orange Solution

Pale Yellow Solution

Ninhydrin test

Blue Solution

Reddish-brown Solution

Xanthoproteic test

1st = Semi cloudy white Solution with powdery ppt 2nd = Yellow-orange Solution

1st = pale yellow solution / no color change occurred 2nd = Light Yellow- orange Solution Violet interphase

Hopkins-Cole test

Violet interphase

Biuret test is a test used to determine the presence of proteins peptide chains. In simpler terms, you can identify if a protein, peptides, or no proteins are present in the solution. Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. The biuret reaction is based on the reduction of Cu2+ to Cu+ which then complexes with the nitrogen atoms on the peptide bonds. The greater the concentration of peptide bonds, the greater the color intensity. When a solution turns blue, we then conclude that there are no proteins present on the solution. With these things as our background, we can now presume that the Base Hydrolyzate has almost no protein or amino acid present in the solution while the intact casein has a lot of proteins in the solution since the produced violet color is darker in shade. Sakaguchi test is a specific qualitative test for the detection of a specific type of protein with the amino acid containing the guanidinium group. This amino acid that contains guanidinium is also known as Arginine. When Arginine is present in a solution tested by Sakaguchi test, Arginine would react to the reagents used by being neutralized and later on when the zwitterions was formed, condense with a-naphthol to produce a red or orange colored solution indicating positive result. Knowing this would imply that the Base Hydrolyzate, which demonstrates a pale yellow solution would have no Arginine present in the compound. The intact casein subject to sakaguchi test would yield a positive result of red or orange colored solution indicating the presence of the amino acid Arginine in the sample.

Ninhydrin test is a test that determines the presence of a-amino acids. The amino acids typically give a blue-purple product, known as Ruhemanns purple. This color change is only specific for the said amino acid. This means that if an amino acid present in the compound does not have an -amino acid, the reagent would create a different coloration with the compound where it was tested. Negative results will have a yellow-orange or red colored solution. From the results that were gathered, we can presume that the Base Hydrolyzate which produced a reddish-brown solution does not have an -amino acid. The Base Hydrolyzate gave a negative result. For the intact casein that yields a blue solution, we can imply that the solution has a lot of -amino acids and this would only mean a positive result. Xanthoproteic test is a reaction due to the presence of phenyl group (-C6H5) in the protein molecules. It involves the nitration of the phenyl rings present in the aromatic amino acids such as tyrosine, phenylalanine, and tryptophan, forming yellow nitrosubstitution products that turn orange when alkali is added. This means that the Base Hydrolyzate that produced a light yellow-orange solution has a very few number of aromatic amino acids. The intact casein that produced a positive result of yellow-orange color would have a lot of aromatic amino acids present on the solution it self. In this case, a positive result. Hopkins-Cole test is a test for the determinacy of the presence of the amino acid tryptophan. When tryptophan is present, this would create a violet ring in between of two different layers. This space in between where result is to be observed is known as the interphase. Both the Casein and the Base Hydrolyzate produced positive results. With this at hand, we can now characterize the types of proteins belonging to both the samples used. Intact casein practically had every amino acid. This is because in all the tests, intact casein gave positive results. Base Hydrolyzate however had only a few positive results. The Base Hydrolyzate only had positive results on Xanthoproteic test and the Hopkins-Cole test. Being positive here means that the Base Hydrolyzate has a Tryptophan amino acid. It is also possible that this is the only amino acid present in the solution for Tryptophan is an aromatic amino acid and yielding positive results on Xanthoproteic test means that the solution had an aromatic amino acid.

EXPERIMENTAL A. Preparation of Protein Suspension: 1. The dried casein was cut to small pieces and was placed in a mortar 2. 15mL of distilled water was added. The mixture was grounded until a fine protein suspension was obtained. B.1. Biuret Test 1 drop of 2.5M NaOH was added to 3 drops of the protein

suspension/hydrolyzate. The mixture was mixed well. 1 drop of 0.01M CuSo4 solution was added. The color was noted B.2. Sakaguchi Test 1 drop of 10% NaOH and 1 drop of 0.02% naphthol solution was added to 5 drops of the protein suspension/hydrolyzate. The mixture was mixed well. 1 drop of 2% NaOBr was added after about 3 mins. The color was noted B.3. Ninhydrin 1mL of water was added to 10 drops of protein suspension. 1mL w/o dilution of water was used for base hydrolyzate. 0.5mL of 0.1% ninhydrin solution was added. The mixture was mixed well. The mixture was heated in boiling water bath for 3 mins. The color was noted. B.4. Xanthoproteic test 1mL of water was added to 10 drops of protein suspension. 1mL w/o dilution of water was used for base hydrolyzate. 3 drops of conc. HN03 was slowly added. The mixture was mixed well and color was noted. Mixture was heated in boiling water bath for 1 minute. The solution was cooled with flowing water. Conc. NaOH was added

slowly drop by drop. The addition of NaOH was continued until the solution was alkaline. The solution was tested with litmus paper. The color was noted B.5. Hopkins-cole test 2mL of Hopkins-cole reagent was added to 2 drops of protein

suspension/hydrolyzate. It was mixed well. The tube was inclined. 2mL of conc. H 2SO4 was added slowly down the side of the tube until two layers was formed. The 2 layers were not disturbed. The color formed at the interphase was noted.

REFERENCES -Menguito, C. A., et. al. (2010). Basic laboratory experiments in biochemistry, Manila Philippines: UST Publishing House -http://www.brilliantbiologystudent.com/biuret_test.html http://www.chemistry.ccsu.edu/glagovich/teaching/316/qualanal/tests/ninhydrin.ht ml -http://bitesizebio.com/articles/doesn%E2%80%99t-play-well-with-others-thechemistry-of-the-autoclave/ -http://faculty.salisbury.edu/~momitchell/Chem_222_files/Ninhydrin%20Test.pdf