ISOLATION AND CHARACTERIZATION OF PROTEINS (GLUTEN ENZYMATIC HYDROLYSIS)

Venneth Pascual, Philina Pasicolan, Paolo Pineda, Christian Pontanres and Christelle Primaleon Group 8 2F Medical Technology General Biochemistry Laboratory

ABSTRACT
Gluten was extracted from dough, a mixture of wheat flour and water. To isolate gluten, the dough was washed under running water to remove the starch content, which is soluble in water thus crude gluten is the insoluble product. Enzymatic hydrolysis, which uses proteases to cut peptide bonds, was performed to yield peptide fragments and some free amino acids and to determine the compositions of the intact protein. Characterization of the intact and hydrolyzed proteins qualitatively used color reactions of different tests and paper chromatography.

INTRODUCTION
Proteins are bio-molecules, essential to all living things due to its diversity and function. They are polymers made up of amino acids, which are joined by peptide bonds, thus a polypeptide. 20 amino acids are presently known to man that can be specifically arranged for a particular protein. All amino acids contain a carboxyl group, amino group and a distinctive Rgroup for each amino acid. The polarity and acid/base property of the R-groups influences the reactions and interactions to other substances. [1] Protein isolation entails the extraction of a single type of protein from a complex mixture. Several factors are considered in the isolation process. This are; (1) 3D structure of the protein (fibrous or globular) (2) Interactions keeping the native conformations (electrostatic, covalent, hydrophobic, H-bonding and Van der Waals) (3) Acid-base property (4) Solubility in different solvents. To isolate, several methods can be used. Isoelectric precipitation for Casein, Heat denaturation for Albumin, Solubility for Gluten and Salt-induced precipitation for Myoglobin. Solubility can be affected by any change on the pH. At the iso-electric point wherein the molecule has a net charge of zero (zwitterion), proteins appear insoluble unionized molecules. Denaturation (unfolding of protein) loses the native conformation of the protein, which has a biological importance is due to factors like; (1) extremities of ph and temperature or (2) exposure to denaturing solvents. [3] Hydrolysis is performed to obtain information about the composition of the intact protein. The products are called hydrolysates. Enzymatic hydrolysis is the breakdown of proteins by cutting the peptide bonds, yielding peptide fragments and some free amino acids by proteases. [6]

Qualitatively, the intact and hydrolyzed proteins can be characterized by the reactions of their side chains, -amino, and -carboxyl groups with different substances producing different colors as a determinant. Paper chromatography is also used wherein the polarity of the protein is the basis for separation. [3] Gluten, a yellowish gray powdery mixture of water-insoluble proteins occurring in wheat and other cereal grains are composed chiefly of the proteins Gliadin and Glutenin. Its presence in flour helps make the production of leavened, or raised, baked goods possible because the chainlike molecules form an elastic network that traps carbon dioxide gas and expands with it. Gluten is also found in special high-protein breakfast foods and other cereal foods and is used in adhesives and as meal for cattle food. [5] The objectives of this experiment are to: (1) analyze chemical groups responsible for color reactions and explain the principle involved in each test (2) perform acid, alkaline and enzymatic hydrolysis on the isolated and enumerate the advantages and disadvantages of each type of hydrolysis (3) To determine the amino acid components of the proteins by paperchromatography.

EXPERIMENTAL A. Compounds tested and samples used

Gluten from wheat flour (intact and enzymatically hydrolyzed), 2% w/v of tryptophan, argininie, proline, cysteine, serine, aspartic acid, tyrosine, histidine, glycine and histidine.

B. Procedure 1. Isolation of Gluten from wheat flour

1 cup of wheat flour was mixed with water to form a dough. The dough was wrapped in a

cheesecloth and washed under running water to remove the starch content. The washings were tested for the presence of starch by treating it with I2 solution. A negative result shows a yellow solution.

2. Enzymatic Hydrolysis of Gluten

1g of crude gluten was mixed with 10 ml of distilled water in a test tube. This protein solution was treated with a meat tenderizer (protease) and covered with cloth. The solution was left to stand overnight under room temperature.

bath. Tested for the evolution of gas by placing a moistened red litmus paper over the mouth of the tube. For Pauly Test, Diazo reagent was prepared by mixing 3-5 drops of 1%sulfanilic acid with 3 drops of 5% NaNO2. 5 drops of the sample and 3-5 drops of 10% Na2CO3 were added to the diazo reagent. Noted the color of the solution.

4. Paper Chromatography
12x15 TLC plate was prepared. The origin was made by making a horizontal line 1.5 cm from the bottom. 13 equidistant points were marked (10 for the standards and 3 for the hyrdolysate samples. Samples were applied on the points (5x for stds. and 10x for hydrolysates) thru capillary tubes with intermittent drying. After the application of samples, the plate was placed in the pre-equilibrated chamber containing the solvent, 1-Butanol:acetic acid:water (4:1:5). The solvents level was below the origin of the plate. The chamber was covered and allowed the solvent to ascend until its level was .5cm from the top edge. Line was marked on the solvent front and the plate was air-dried and sprayed lightly with 1% ninhydrin reagent. The plate was then placed in the oven for 1-3 minutes. Spots were encircled. Calculate for the Rf values of the standards and hydrolysates.

3. Qualitative Color Reactions

For all tests, .5g of protein was mixed in 1ml of distilled water (intact) and .5ml of hydrolyzed (enzymatic) solution was used. For Biuret test, 20 drops of 2.5M NaOH was added to the sample and mixed then added 2-3 drops of .1MCuSO4 solution then shaken. Noted the color of the solution. For Ninhydrin test, 6-10 drops of 1% ninhydrin solution was added to the samples then heated in a boiling test tube. Noted the color of the solution. For Xanthoproteic test, 10 drops of conc. HNO3 was slowly added to the samples. Noted the color change. 10 drops of conc. NaOH was then slowly added then mixed. Noted the color of solution again. For Millons Test, 5 drops of Millons reagent was added to samples. Noted the color of the solution. For Hopkins-Cole Test, 20 drops of HopkinsCole reagent was slowly added then mixed. Test tube was inclined and then slowly added 20 drops of conc. H2SO4. Tube was not shaken. Noted the color of the solution. For Sakaguchi test, 10 drops of 10% NaOH and 10 drops of .02% napthol solution were added to the samples then mixed. Tube was stood for 3 minutes. 3 drops of 2% NaOBr was added. Tube was mixed and noted the color of the solution. For Nitroprusside test, .5ml of 3M NaOH was added to .5ml of sample and .25ml of 2% nitroprusside solution. Noted the color of the solution. For Fohls test, 5 drops of 30% NaOH to 10 drops and 2 drops of 5% (CH3COOH)2Pb were added to the samples then placed in a boiling water bath. Noted the color of the solution. Test for amides. 1ml of 20% NaOH was added to 10 drops of sample place in a boiling water

RESULTS AND DISCUSSION 1. Isolation

Differences in solubility were the principle used to isolate gluten from dough. Because starch, a component of dough, is soluble in water, washing the dough in cheesecloth under running water can isolate gluten. The washings were tested with I2 solution to check the presence of starch. A positive result gives a blue-black solution and a negative result gives a yellow solution. Washing with water must be done until a negative result is obtained. Crude gluten is insoluble in water therefore, the product.

2. Hydrolysis (enzyme)
To examine the compositions found in the intact protein, hydrolysis is done. Enzymatic hydrolysis uses proteases, which are found naturally in all organisms and catalyze the hydrolytic cleavage of peptide bonds giving peptide fragments and some free amino acids.

3. Color reactions
Test Biuret Ninydrin Xanthoprteic Millons Hopkins-Cole Sakaguchi Nitroprusside Fohls Test for amide Pauly Intact Blue (+) Blue-violet (+) Orange (+) Colorless (-) Purple (+) Turbid soln (-) Yellow (-) Black (+) Red to blue (+) Red (+) Enzymatic hydrolysate Blue (+) Violet (+) Turbid soln. (-) Turbid soln. (-) Turbid soln. (-) Turbid soln. (-) Yellow (-) Turbin soln. (-) Red to blue (+) Red-orange (+)

Table 1. Results for Qualitative Color Reactions Every test has different principles, mechanisms and concepts. Results varied to every test due to the unique characteristics of the samples in the reactions with its side chains, -amino and carboxyl groups and the reagents used for every test. Biuret test is a general test used to determine the presence of peptide bonds in proteins. It is a complexation reaction. Intact proteins should only be the one producing a positive result because its peptide bonds have not been broken unlike the hydrolyzed samples. Ninhydrin test determines the presence of amino groups and its principle is oxidative deamination and decarboxylation. Proline should only yield a negative result. A blue-violet solution indicates a positive result. Both samples yielded a positive result and indicated the presence of amino groups. Xanthoproteic test detects the presence of aromatic rings. Nitration of the aromatic ring is its principle. Amino acids like tryptohan and tyrosine would yield a positive result although phenylalanine has an aromatic ring; it is inactive thus giving a negative result. A positive result is seen as yellow solution upon addition of a HNO3 and turning to orange upon addition of NaOH. Millons test checks for the presence of Tyrosine. Complexation between the phenolic group and mercury located in the reagent is the principle of the reaction. A red precipitate would yield a positive result.

Hopkins-Cole Test checks for the presence for Tryptophan. Condesation of the indole group with glyoxylic acid H2SO4 is the principle. A purple interphase between the two immiscible layers indicates a positive result. Sakaguchi Test checks for the presence of free or intact Arginine. Reaction among the Guanidino group, naphthol and oxidizing agent is the principle. Basic hydrolysis destroys arginine, producing ornithine and urea, therefore should give a negative result. A positive result is indicated by a red solution. Nitroprusside checks for the presence of Cysteine, which is partially destroyed in the test. Complexation is the principle. A red solution shows a positive result. Fohls Test checks for the presence of sulfercontaining proteins and amino acids; Cysteine and Methionine. Fusion then ionic interaction is the principle. A black solution or precipitate is the indicator for a positive result. Test for amides seeks for the presence of primary, secondary, tertiary amides and nitriles. A change from red to blue of the litmus paper yields a positive result. Both samples had positive results indicating the presence of amides. Pauly test detects for the presence of Tryptophan, Tyrosine and Histidine. Its principle is diazotization. Diazo reagent, which is composed of sulphanilic acid dissolved in HCl mixed with Sodium Nitrile and HCl, forms the diazo salt/reagent. Reaction between the salt and either of the three amino acids gives a red solution (azo dye).

4. Paper chromatography
Standards Tryptophan Arginine Proline Cysteine Serine Aspartic Acid Tyrosine Histidine Glycine Alanine Rf values 0.42 0.12 0.31 0.10 0.19 0.21 0.40 0.11 0.20 0.34

* no results obtained from hydrolysates

Table 2. Rf values of Standards used

The polarity of the standards and samples are the main basis for the differences in the distances traveled in the chromatogram. The solvent Butanol:Acetic acid: water (4:1:5) is more nonpolar than polor due to the large ratio of butanol therefore, the longer the distance traveled by the standards/sample = stronger affinity to the mobile phase = higher the rf value = the more non-polar. Due to many errors committed, the group sample was discarded and data was obtained from other groups as instructed by the teacher.

REFERENCES
From books: [1] Campbell, M.K. and Farrell, S.O. (2012). Biochemistry. 7th ed. International editon. Brooks/Cole CENGAGE learning. [2] Cox, M.M. and Nelson D.L. (2000). Lehninger Principles of Biochemistry. 3rd ed. New York: Worth Publishers. [3] Crisostomo, A.C., Daya, M.L., De Guia, R.M., Farrow, F.L., Gabona, M.G., Liu, M.I., et al. (2010). Laboratory Manual in General Biochemistry. C & E Publishing, Inc. From the internet: [4] Del Rosario, M.A. Isolation and Characterization of Proteins. http://www.scribd.com/doc/49052097/Isolationand-Characterization-of-Proteins retrieved: 12/16/12 [5] Gluten defined. http://www.britannica.com/EBchecked/topic/235 984/gluten retrived: 12/16/12 [6] Enzymatic Hydrolysis. http://www.biologyonline.org/dictionary/Enzymatic_hydrolysis retrieved: 12/16/12 [7] Qualitative Analysis of amino acids. http://amrita.vlab.co.in/?sub=3&brch=63&sim=1 094&cnt=1 retrieved: 12/20/12