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Isolation, Qualitative Color Reaction and Alkaline Hydrolysis of Intact Protein Gluten

V.R. Acosta, J. Alcala, S.A. Aliola, M.C. Azarcon, Z. Baccay, C. Beleno 2G Pharamacy Faculty of Pharmacy, UST

Abstract
Chemical analysis can be either qualitative or quantitative in nature. In qualitative analysis we work to identify the substances present in a given sample. We are not concerned with the quantity of each substance, but only whether certain substances are present or absent. On the other hand, in quantitative analysis we are concerned with determining the amount of each component present in a sample. For example, those of you who took Chemistry 1A at Foothill College should remember that we prepared and then analyzed the green crystals, K3Fe(C2O4)3(H2O)3. Qualitative analysis of this ioniccompound could be used to reveal that iron, potassium and oxalate ions are present. It could also be used to determine that the iron is in its +3 oxidation state and that the substance is a hydrate. On the other hand, quantitative analysis of the green crystals would reveal that it contains 11.4% Fe3+, 23.9% K+, 53.7% C2O42and 11.0% H2O by mass.

Introduction
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded 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 selenocysteineand in certain archaeapyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes. If the R- group contains a carboxyl group, the amino acid displays acidic properties. When an amino group is found instead, a basic amino acid is the result and finally, alkyl groups containing only carbon atoms or those with alcohol or sulfur group for the R-group constitute a neutral amino acid. In these structures is an internal acid-base reaction can occur as a resulting in formation of a zwitterions, an internal salt, dipolar ion and is a compound that contains both a positive and a negative charge.

Figure2. Structure of a zwitterions

Figure1. General structure of an amino acid

Proteins have precise three-dimensional configuration, primary structure which refers to its amino acid sequence which actually determines the protein shape, secondary structure which frequently contain helix and pleated sheet structures, tertiary structure comprise of the further interaction of the backbone of a protein, it is the folding and coiling of a peptide chain to produce the complex, rather rigid over-all conformation and lastly, quaternary structure refers to the interaction between subunits of a protein. Those proteins that have only one subunit do not possess a quaternary structure. The glue that holds

the shape together mayconsist of hydrogen bonds, disulfide bonds, hydrophobic interaction, ionic and covalentbonds, electrostatic links and Van der Waals. Many of these interactions take place betweenside chain or R-groups of amino acid and carboxyl group. Hydrolysis of proteins refers to the breaking of peptide bonds that link amino acids together. The final result is the amino acid that makes up the protein.

1.Biuret Test
In the prepared test tube with the intact protein solution, 20 drops of 2.5 M NaOH was added and mixed well. Another 2-3drops of 0.1 M CuSO4 solution was added. The test tube was shaken and the color of the solution was noted.

2.Ninhydrin Test
The sample was treated with 6 - 10 drops of 0.1% ninhydrin solution and was heated in a boiling water bath. The color of the solution was noted.

The 20 amino acid commonly found as hydrolysis product of protein contain -carboxyl group, an amino group and a distinctive R-group substituted on a -carbon atom. It is done to obtain information about their composition and can be carried out by treating protein with acid, alkali, or proteolytic enzymes. The protein that was isolated and was used totest for determination of structure, functionalgroup, and aromatic and amide side chain isgluten isolated from source which is wheatflour.Gluten can be defined as the rubbery mass thatremains when wheat dough is washed toremove starch granules and watersolubleconstituents. It is a mixture of proteins notreadily soluble in water that occurs in wheatand most other cereal grains. Its presence inflour make production of leavened baked goodspossible because the chain-like glutenmolecules form elastic networks that trapscarbon dioxide gas and expands with it.

3.Xanthoproteic Test
In the prepared test tube with the intact protein solution, 10 drops of concentrated HNO3 was slowly added and was mixed. The color of the solution was noted. Slowly added after was 10 drops of concentrated NaOH and was mixed well and the color of the solution is again noted.

4.Millons Test
The sample was treated with 5 drops of Millons reagent and the change in color was noted.

5.Hopkins - Cole Test


For Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was slowly added to the sample protein and was mixed well. The test tube was inclined and 20 drops of concentrated H2SO4 was slowly added along the side. The color and the interface were noted.

Materials and Method


A.Isolation of Gluten
In isolating Gluten, one cup of wheat flour,cheesecloth and Iodine solution was required.The one cup of wheat flour was added with water until the dough turns thick. The dough was wrapped in cheese cloth and was placed under running water until all starch was removed. The washings were tested for presence of starch by adding Iodine solution and were repeatedly done until a negative result was obtained. The remaining insoluble material in the cheesecloth is the crude gluten.

6.Sakaguchi Test
The sample was treated with 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution and was mixed. It was left to stand for 3 minutes, after 3 drops of 2% NaOBr was added and was mixed. The color produced was noted.

7.Nitroprusside Test
In the prepared test tube with the intact protein solution, 0.5 ml of 3 M NaOH was added. Another 0.25 ml of 2% Nitroprusside solution was placed. The decolorization of the solution was noted.

B. Qualitative Color Reaction of Gluten


For each test, a separate test tube with 0.5grams of intact protein and 1 ml of distilled water was prepared.

8.Fohls Test
Five drops of 30% NaOH and 2 drops of 5%(CH3COO)2Pb was added to the intact protein solution. After which, the test tube was placed in a boiling water bath and theappearance of sediment was noted.

9. Test for Amides


Ten drops of the protein sample solutionwas treated with 1ml of 20%NaOH. It wasplaced in a boiling water bath after. Theevolution of gas was tested during heatingby placing a moistened red litmus paperover the mouth of the tube. The resultswere noted.

C. Alkaline Hydrolysis of Gluten


To hydrolyze the intact protein which is gluten,10 ml of 4 M NaOH was added to 0.5 g isolated protein and was placed in a hard glass test tube and was labeled. The tube was covered with cotton and was submitted for autoclaving in15psi for 5 hours after which the appearance was noted and 10 ml of distilled water wasadded and mixed well. The mixture wastransferred into a 250ml beaker. The mixturewas neutralized with 1M HCl and thishydrolysate was used for another set of characterization test and chromatography.

Tests Biuret Ninhyfrin Xantoproteic Millons Hopkins Cole Sakaguchi Nitroprusside

Color Reaction Blue Violet Soln Blue Violet Soln Clear Yellow Soln Peach Soln Clear Violet ring Clear Yellow Soln Clear Yellow Soln & Brown Sediments Fohls Black ppt Test for Amides Red Blue Litmus Paper
Table1. Results of Color Reaction of Intact Protein Solution

1. Biuret Test ( test for peptide bonds)


The Biuret test is a positive test for proteins but not for amino acid. The evidence for the test consists of formation of a violet-pink complex when cupric ion, in basic solution is added to any polymer such as protein which contains multiple amide bonds. A bluecolored solution indicates a negative test or those fewer than two peptide bonds are present.

Results and Discussion


A. Isolation of Gluten
The isolated protein, Gluten was cream colored,sticky, elastic, gum-like texture and smells likeof oatmeal. Wheat flour has two major components: crudegluten and starch. To separate the gluten whichis the intact protein from starch, a separationtechnique is utilized, the best for which wasdifference in solubility. Starch, a white odorlesspowder carbohydrate which is a chief storageform of carbohydrates in plant is soluble inwater while gluten, on the other hand, is anelastic mixture composition of glutenins andgliadins which is insoluble in water. Considered in the isolation of an intact proteinfrom its sources are 3-Dimensional structure of protein, the interaction that keeps the nativeconformation of the protein functional, its acid-base property and its solubility in proteins.

2. Ninhydrin Test( test for amino acid)


The ninhydrin test is positive for amino acid and some proteins. Free amino acids can react with ninhydrin reagent yielding a deep purple solution. Most of amino acids have free amino groups Gluten is positive for this test.

3. Xanthoproteic test (test for aromatic side chain)


The test depends upon a reaction with a specific type of amino acid chain. Aromatic rings often have the ability to undergo nitration reaction, which is the addition of NO2 group to the ring that is why it is a positive test for side chains in tyrosine and tryptophan. With these amino acids present, addition of nitric acid and heat will result in a yellowishcolored solution. Upon addition of base, color will change to orange. Gluten is positive of aromatic sidechain.

B. Qualitative Color reaction of Gluten


Amino acids have a variety of chemically reactive groups that can be used to characterizeboth free amino acid and proteins. The following tests are used to detect presence of amino acid and proteins and distinguish between them.

4. Millons Test (test for phenolic groupcontaining side chain)


The color produced in Millon's test is given by derivatives of benzene in which hydrogen in the ring has been replaced by a hydroxyl group. The reaction serves as a test for the presence of tyrosine. Gluten sample showed negative result.

5. Hopkins-Cole Test (test for amino acid tryptophan)


The clear violet ring produced is due to the formation of a compound from the glyoxylic acid in the reagent and the tryptophan in the protein. A similar color is produced when sulphuric acid is added to a protein solution in the presence of a trace of formaldehyde. Gluten showed a positive result for the presence of tryptophan.

References
http://www.foothill.edu/attach/ps me/sinha.QualDiscussion.pdf 2. http://en.wikipedia.org/wiki/Protei n
1.

6. Sakaguchi Test (test for the guanidinogroup of arginine)


In basic conditions, alpha naphthol and sodium hypobromite/chlorite react with the amino acid containing guanidine group to form red-orange complexes. Gluten sample showed negative result thus, does not consist of arginine aminoacid.

7. Nitroprusside Test (test for free SH group)


Amino acid with free thiol groups due to cysteine yields a positive result, red to red violet decolorization when introduced to Nitroprusside. Gluten yields a positive result for this test.

8. Fohls Test (test for sulfur containing amino acid)


This reaction is used for determination of Scontaining amino acids. Heating of protein solution in an alkaline medium leads to the formation of sulfide (Na2S) if the protein contained sulfur amino acids such as cysteine, cystine or methionine. A positive result is red to red violet decolorization. Further reaction of Na2S will lead acetate a dark brown colored precipitate is formed

9. Test for Amides (test for the presence of Asparagine and Glutamine)
The red litmus paper turned blue indicates a basic component of the gluten, thus gluten is positive for the presence of a basic amino acid. From the data gathered, Gluten was chemically determined to be a basic amino acid contain sulfide and peptide bonds, has an aromatic side chain except for tyrosine. It is also positive for the presence of disulfide bond due to cysteine

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