ISOLATION AND COLOR REACTION OF INTACT PROTEIN (GLUTEN)

Kriziaoumo P. Orpia, Michael Gavin G. Quinto, Nico Joy C. RidulmeKathryn Chemaine L. Samorano and Ryza Ruba

Group 8 2E Medical Technology Biochemistry Laboratory

ABSTRACTGluten, the intact protein, was separated from wheat flour by dissolving the starch that accompanied it in the process of making wheat flour. The intact protein was tested to describe its structure and functional groups. Such test used were to predetermine the presence for amides and aromatic side chains

INTRODUCTIONGluten is a mixture of proteins not readily

soluble in water that occurs in wheat and most other cereal grains. Its presence in flour makes production of leavened baked goods possible because the chain-like gluten molecules form elastic network that traps carbon dioxide gas and expands with it [4]. Gluten in the experiment is taken from wheat flour. The objectives of this experiment are to isolate gluten from the flour mixture and to analyze and determine the chemical group responsible for the color reactions and explain the principle involved in each test.

EXPERIMENTALA. Materials and Compounds Used

1. For the Isolation of Gluten Wheat flour Water Cheesecloth Iodine Solution

2. For the Qualitative Color Reactions 2.5M Sodium Hydroxide (NaOH) 0.1M Copper Sulfate (CuSO4) Millon’s Reagent Hopkins-Cole Reagent 10% Sodium Hydroxide (NaOH) 0.1% Ninhydrin Solution 3M Sodium Hydroxide (NaOH) 2% Nitroprusside Solution 30% Sodium Hydroxide (NaOH) 1% Sulfosalicylic Acid 10% Sodium Carbonate (Na2CO3) Conc. Sodium Hydroxide (NaOH)

solution 0.02% Naphthol Solution Conc. Nitric Acid (HNO3) Conc. Sulfuric Acid (H2SO4) 6M Sodium Hydroxide (NaOH) 2% NaOBr 20% Sodium Hydroxide (NaOH) Hot plate Beaker 5% Lead Acetate (Pb(CH3COO)2)a. 5% Sodium Nitrite (NaNO2)

B. Procedure1. Isolation of Gluten

In isolating gluten, one cup of wheat flour is washed with water. Continue to add water until the dough turns thick. As the dough thickens, wrap it onto the cheesecloth and place it in running water. This way starch is removed from the dough. The procedure is done until such time all the starch from the dough is removed. To test whether starch is still present, gather water droppings below the dough in a test tube and use iodine solution. If the iodine solution is negative, it can be safe to assume that all starch is removed from the dough. The remaining insoluble material inside the cheesecloth is your crude gluten.

2. Qualitative Color Reaction of GlutenThe test here aids to identify the chemical

groups that are bonded to the -carbon. In ten test tubes, 0.5g of gluten is dissolved in 1 mL distilled water. Each test tube corresponds to a particular test. Label each test tube according to their corresponding test to avoid confusion.

a. Biuret TestFor the biuret test, treat the protein solution with 20 drops of 2.5M NaOH and mix well. Next step is by adding 2-3 drops of 0.1M CuSO4 solution. Take note of the color of the gluten solution after treated.

b. Ninhydrin TestFor the Ninhyrin Test, treat the diluted sample with 6-10 drops of 0.1% Ninhydrin solution then proceed by placing the test tube containing the sample in a water bath and observe the appearance of a blue violet coloration.

c. Xanthoproteic TestIn Xanthoproteic Test, the protein solution is slowly treated with 10 drops concentrated Nitric Acid (HNO3). Observe caution for handling acids. Before proceeding to the next step, take note of the color of the solution. Next slowly add

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10 drops of Sodium Hydroxide (NaOH) and again note the color of the sample.

d. Millon’s TestThe sample is treated with 5 drops of Millon’s reagent and be noted for its color reaction.

e. Hopkins-Cole TestSlowly treat the gluten sample with 20 drops Hopkins-cole reagent and mix well. Then incline the tube add slowly along the side 20 drops of concentrated sulfuric acid. (H2SO4). Observe caution. Note the color of the sample.

f. Sakaguchi TestThe gluten sample is treated with 10% Sodium Hydroxide (NaOH) and 10 drops of 0.02% Naphthol Solution. Let the sample stand for 3 minutes and then add 3 drops 2% NaOBr. Again note the color of the solution.

g. Nitroprusside TestFor the nitroprusside test, add 0.5mL 3M of Sodium Hydroxide (NaOH) and 0.25 mL 2% Nitroprusside Solution. Note the change in color to a red solution.

h. Fohl’s TestAdd 5 drops of 30% Sodium Hydroxide (NaOH) and 2 drops of Lead Acetate (Pb(CH3COO)2) to the sample then proceed by placing it in a water bath. Note for the appearance of dark or brown sediments

i. Test for AmidesAdd 1mL 20% NaOH to 10 drops of the gluten sample then proceed by placing it in the water bath. Test for the evolution of gas by placing moisten red and blue litmus paper over th mouth of the tube.

j. Pauly TestFirst step is by preparing a diazo reagent by mixing 3-5 drops 1% sulfosalicylic acid with 3 drops of 5% Sodium Nitrite (NaNO3). Add 5 drops of the sample and 3-5 drops of 10% Sodium Carbonate (Na2CO3) to the diazo reagent. Note the appearance of the red coloration.

RESULTS AND DISCUSSION1. Isolation of Gluten

Crude Gluten was obtained by washing the dough to the water. The crude gluten obtained was from wheat flour, an ingredient to produce

bread particularly leavened bread. Wheat flour has two major composition, starch and crude gluten. In order to separate gluten from starch, certain separation techniques must me used to separate the two components. The best way to separate the two is by means of solubility.

Starch is a white odorless powdery carbohydrate (C6H10O5)x which is a chief storage form of carbohydrates in plants [2]. Starch is soluble in water while gluten is insoluble. Hence gluten can be separated by using the solubility rule.

Gluten is an elastic mixture composite of proteins glutenin and gliadin. Gluten has chemical groups that make up its characteristics. Gluten undergoes a series of Qualitative analysis to further understand the structure of gluten.

2. Qualitative Color Reaction of GlutenIn table 1, the results of the color reactions

are tabulated and noted. 0.5g of gluten was placed in ten test tubes together with 1mL of Distilled water.

TABLE 1. RESULTS OF COLOR REACTION OF GLUTEN

COLOR REACTION TEST OBSERVATION/SBIURET TEST Violet

NINHYDRIN TEST Blue VioletXANTHOPROTEIC TEST Yellow

MILLON’S TEST WhiteHOPKIN’S-COLE TEST Violet (interface)

SAKAGUCHI TEST Light YellowNITROPRUSSIDE TEST Red

FOHL’S TEST Brown precipitateTEST FOR AMIDES R-B (Basic)

PAULY TEST Pale Yellow

a. BIURET TEST (Test for Peptide Bonds)

The biuret test, it is basically the most fundamental test for proteins, a general test for proteins [1]. The test is used for the presence of peptide bonds. When a protein synthesizes with Copper (II) Sulfate, test will confirm a protein is present in an unknown forming a protein-copper complex which has a violet coloration.

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b. NINHYDRIN TEST (test for Free NH2)Amino acids with a free –NH2 group and proteins containing free amino groups react with ninhydrin to give a purple-blue complex. Gluten resulted positive to the test.

Illustration from: Laboratory Experiments for Organic and Biochemistry

c. XANTHOPROTEIC TEST (test for Aromatic Side Chains)The Gluten sample reacted to the concentrated Nitric Acid (HNO3) forming a yellow substance or aromatic nitro compound [5]. After adding an alkali or a strong base in the form of Sodium Hydroxide, the color shifted to orange. Such amino acids like tyrosine, phenylalanine and tryphtophan. The gluten sample is positive for aromatic side chains.

d. MILLON’S TEST (test for phenolic group containing amino acids)

The Millon’s test is the test to determine the presence of Tyrosine [5], which is the amino acid that contains a phenolic ring, but the gluten sample which was tested for this test showed negative result. The gluten contains side aromatic chains except tyrosine.

e. HOPKINS-COLE TEST (test for the amino acid tryphtophan)

The gluten sample was first treated with Hopkins-Cole reagent and then slowly added concentrated Sulfuric Acid (H2SO4). The visible result would be the color at the interface of the two substances. The interface is colored violet. The positive result of Hopkins-Cole test is a violet color. This violet color is due to the presence of the indol group. [5] Gluten is positive with tryptophan

f. SAKAGUCHI TEST (Test for the guanidino group of Arginine)

The Sakaguchi test is a test for the presence of the guanidine group specifically found in Arginine [5]. The gluten sample however test for a negative results which generally means the gluten sample does not consist if a Arginine amino acid.

g. NITROPRUSSIDE TEST (test for free –SH group)

The test results to a red color or red violet. Such result can be seen in Cysteine. Therefore it can be stated that the amino acids with free thiol groups due to cystiene yields a red color when introduced to Nitroprusside’s or Bollin’s Test [5]. Gluten was positive for the results.

h. FOHL’S TEST (test for sulfur containing amino acids)

The Fohl’s test shown gluten was positive for the brown precipitate. Fohl’s test is the same in test as to nitroprusside test. They are the test to determine is the protein contains sulfur bound compounds.

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Illustration from : Laboratory Experiments for Organic and Biochemistry

i. TEST FOR AMIDES (test for the presence of Asparagine and Glutamine)

The gluten solution was treated with Sodium Hydroxide and was hot bathed until evolution of gas occurs. Using litmus paper, the red litmus paper turned blue. This indicates a basic component of gluten. Gluten is positive for the presence of basic Amino Acids

j. PAULY TEST (Test for Histidine and Tyrosine)

The gluten sample showed negative result to the Pauly test and denies the presence of Histidine. The diazo Reagent would react to the presence of amino immidazole and phenol to form a colourful azo compound (deep red) [5].

The gluten in general is a mixture of protein consisting of glutenin and gliadin. From the results gathered from the color reaction, Gluten can be chemical described as an amino acid that contains sulfide bonds, peptide bonds. Some chains between each carbon are also examined. Gluten has an aromatic side chain except from tyrosine. Gluten was positive for the presence of basic amino acids. It was also positive for the presence of disulfide bond due to cysteine. REFERENCES

From Books[1] Bettelheim, Frederick A., Landersberg

Joseph M. (2001), Laboratory Experiments for Organic and Biochemistry Philadelphia: Harccourt College Publishers. P.201

[2] Merriam-Webster’s Medical Dictionary (2006). Springfield Massachusetts: Merriam-Webster Incorporated

[3] Copeland, Robert.(1994). Methods of Protein Analysis NewYork: Chapman & Hall,p.46

[4] Gluten, Britannica Concise Encyclopedia (2002) U.S.A.: Encyclopædia Britannica

From the Internet[5] Undergraduate First Year Practical Manual

http://www.scribd.com/doc/183215/UNDERGRADUATE-FIRST-YEAR-PRACTICALS-MANUAL

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