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Protein Isolation and Characterization Tabaong, Mara Danielle; Tejada, Rolando; Ventura, Raymond; Villanueva, Marie Antoinett; Villegas,

Katrina; Vinluan, Kenna Group 8 2DPH BIOCHEMISTRY LABORATORY Abstract: The intact protein was isolated from different sources by isoelectric precipitation, difference in solubility and salt-induced precipitation. Casein was isolated from skimmed milk, gluten from wheat flour and myoglobin from beef. The intact proteins were also characterized by colorimetric reactions such as Biuret, Ninhydrin, Xanthoproteic, Millons, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohls, and Amide Tests. The Biuret Test was done by adding 20 drops of 2.5 M NaOH and 2-3 drops of 0.1 M CuSO4 solution. The Ninhydrin Test was done by placing 6-10 drops of 0.1% ninhydrin solution and by heating a boiling water bath. The Xanthoproteic Test was done by adding 10 drops of conc. HNO3 and 10 drops of conc. NaOH. The Millons Test was done by adding 5 drops of Millons reagent. The Hopkins-Cole Test was done by adding 20 drops of Hopkins-Cole reagent and 20 drops conc. H2SO4. The Sakaguchi Test was done by adding 10 drops each of 10% NaOH and 0.02% naphthol solution and 3 drops 2% NaOBr. The Nitroprusside Test was done by adding 0.5 mL of 3 M NaOH and 0.25 mL 2% nitroprusside solution. The Fohls Test was done by adding 5 drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb and placing into a boiling water bath. Lastly, the Test for Amides was done by adding 1 mL of 20% NaOH and placing in water bath. All the intact proteins showed a positive result: violet solution for Biuret Test, blue-violet solution for Ninhydrin Test, orange solution for Xanthoproteic Test, flesh ppt. for Millons Test, violet ring at the interface for Hopkins-Cole Test, red-orange solution for Sakaguchi Test, yellow solution for Nitroprusside Test, black ppt. for Fohls Test, and red to blue litmus paper and yellow-orange solution for Test for Amides. Introduction: A protein molecule is a long chain of amino acids linked by peptide bonds. The properties are determined by the order or sequence of the amino acids in its molecule, and by the threedimensional structure of the molecular chain. The chain folds and twists and then forming its conformational structure which gives its distinctive properties [1]. Proteins are large, complex, biologically-important molecules composed of amino acids joined by peptide bonds. The number of amino acids used in a single protein can be many hundreds. Proteins are essential to all living organisms. As enzymes, they regulate all aspects of metabolism. Structural proteins such as keratin and collagen make up the skin, claws, bones, tendons, and ligaments; muscle proteins produce movement; hemoglobin transports oxygen; and membrane proteins regulate the movement of substances into and out of cells. For humans, protein is an essential part of the diet, and is found in greatest quantity in soy beans and other grain legumes, meat, eggs, and cheese. During digestion, protein molecules are broken down into amino acids which are then easily absorbed into the body [1]. Specific reactions are used for the purpose of identifying amino acids and proteins in biological media, for qualitative and quantitative analysis. Biuret test is used to determine peptide

bonds [2]. Ninhydrin test is typical for amino acids [3]. Xanthroproteic test is a test for the detection of aromatic proteins in which concentrated nitric acid reacts with the proteins to form a yellow color that is intensified to orange-yellow by the addition of alkali [4]. Millons test is used to demonstrate the presence of the amino acid tyrosine [5]. HopkinsCole test is specific for tryptophan group [6]. Sakaguchi test is a test for guanidines, i.e arginine and peptides that contain it [7]. Nitroprusside test is a test for cystinuria. Fohls test is used to know if sulfur-containing amino acids are present [8]. Test for amides is used to detect R-groups of asparagines and glutamine. The objectives of this experiment are (1) to isolate casein from skimmed milk by isoelectric precipitation, gluten from wheat flour by difference in solubility, and myoglobin from beef by salt-induced precipitation; and (2) to analyze chemical groups responsible for color reactions and explain the principle involved in each test. Experimental: Isolation of Casein from Skimmed Milk Powdered non-fat milk with a weight of 2.0 grams and 50.0 ml of water was placed into a 100-ml beaker. The mixture was then heated in a temperature of about 400 C. A 10% acetic acid was added dropwise when the mixture has reached 400 C and then stirred gently after every 5 drops. Acetic acid was added continuously until the pH reaches 4.6. The congealed casein was filtered by using gravity filtration and then the decantate was set aside for the isolation of albumin. The casein residue was dried then its weight % was calculated.

Isolation of Gluten from Wheat Flour Water was added to 1 cup of wheat flour to make thick dough. The dough was wrapped in the cheesecloth then placed under running water until all starch is removed. The washings was tested with I2 solution until a negative results is obtained. The insoluble material was the crude gluten. Isolation of Myoglobin from muscle A 6.0 g minced beef was placed with 6 ml 70 % (NH4)2SO4 solution in a small beaker. The mixture was gently stirred for 1 minute to release the myoglobin. The dark-red extract was expressed into a new beaker using cheesecloth. The extract was centrifuged at 13 000 x g for 15 minutes. A 1.5 ml of supernatant was transferred into another empty centrifuge tube. A ~0.30-0.35 of (NH4)2SO4 crystals ground was added to fine powder then mixed gently until the solid dissolves. The samples were centrifuged again for at least 5 minutes. The supernatant was decanted off and then the appearance of the purified myoglobin residue was described. Qualitative Color Reactions of the Intact Proteins The intact proteins were tested with different characterization tests namely: Biuret, Ninhydrin, Xanthoproteic, Millons, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohls and Amide. There were 9 test tubes prepared for each of the test reaction. Each test tube consisted of 0.5 g of intact protein in 1 mL distilled water. In Biuret Test, 20 drops of 2.5 M NaOH and 2-3 drops of 0.1 M CuSO4 solution were added to the samples. Afterwards, they were shaken and then observed for color changes. For Ninhydrin test, 6-10 drops of 0.1 % Ninhydrin solution was added to the

diluted sample and heated in a boiling water bath. The appearance of a blueviolet coloration was taken note of. In Xanthroproteic test, concentrated nitric acid and concentrated sodium hydroxide, 10 drops each, were added slowly and then mixed. Color changes after each addition were observed. In Millons test, 5 drops of Millons reagent was added to the diluted sample with the color taken note of. For Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was slowly added to the sample and mixed well. The test tube was inclined to add slowly 20 drops of concentrated H2SO4. The color at the interface was noted. In Sakaguchi test, 10 drops each of 10% NaOH and 0.02 % -naphthol solution was added to the samples then mixed and was let stand for 3 minutes. Afterwards, 3 drops of 10% NaOBr was added. It was then mixed and color change was noted. In Nitroprusside Test, 0.5 ml of 3M NaOH and 0.25 ml 2 % nitroprusside solution was added. Formation of a red solution was noted. In Fohls Test, 5 drops of 30 % NaOH and 2 drops of 5% (CH3COO)2Pb were added to the samples then placed in a water bath. Appearance of black precipitate was noted. Lastly, a test for aide was made by adding 1 ml of 20 % NaOH to 10 drops of the sample then placed in a boiling water bath. Evolution of gas during heating was tested by placing a moistened red litmus paper over the mouth of the test tube and result was noted. Results and Discussions: Isolation of Proteins (a) casein from skimmed milk Casein was isolated from the skimmed milk by isoelectric precipitation. All proteins have an isoelectric point, pI, a pH at which they have no net charge, and they are least soluble at their pI

because there are no net electrostatic repulsions between protein molecules. So to isolate casein at its isoelectric pH, an acid is used to adjust the pH to 4.6. (b) gluten from wheat flour Gluten was isolated from the wheat flour by solubility differences. This was done by washing the dough with water. This removes the starch; the insoluble material is the gluten. (c) myoglobin from beef Myoglobin was isolated from the beef by salt-induced precipitation wherein the proteins are less soluble at salt concentrations (high ionic strength) because the salt ions bind most of the water molecules. Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract. Qualitative Color Reactions of the Intact Proteins
Table 1. Results obtained from the tests

Color Reaction Biuret Ninhydrin Xanthoproteic Millons Hopkins-Cole Sakaguchi Nitroprusside Fohls Test for Amide

Intact Protein (casein, gluten, myoglobin) violet solution blue-violet solution orange solution flesh ppt. violet ring at the interface red-orange solution yellow solution black ppt red to blue litmus paper; yellow-orange solution

The Biuret Test for proteins positively identifies the presence of proteins in solution with violet color. Biuret reacts with copper (II) ions in a basic solution to form a violet complex.

The peptide linkages in proteins resemble those in biuret and also form deep violet complexes with basic copper (II) ions in solution. The Ninhydrin Test showed a positive result of blue-violet solution. It is because proteins also contain free amino groups on the alpha-carbon and can react with ninhydrin to produce the blue-violet color. The Xanthoproteic Test showed a positive result of orange solution. The principle around this is the nitration of aromatic rings via SEAR. The Millons Test showed a positive result of flesh precipitate. In this test, the phenol group of the tyrosine was nitrated by nitric acid. The nitrated tyrosine complexes mercury (I) and mercury (II) ions into the solution to form old rose/flesh to red precipitate. So proteins with tyrosine will show a positive result. The Hopkins-Cole Test showed a positive result of violet ring at the interface. The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet ring product. The Sakaguchi Test has a (+) result of red to red-orange color. The principle around this is about Complexation (base-catalyzed condensation of -naphthol with the guanido group of Arginine) The Nitroprusside Test showed a positive result of yellow solution because the cysteine group reacts with nitroprusside in alkaline solution. The Fohls Test (Lead (II) acetate Test) has a (+) result of brown to black precipitate. The principle is about the

degradation and substitution reaction to form PbS. The Test for Amide showed a positive result of yellow-orange solution and the red litmus paper turned into blue.

Figure 1. Colorimetric Results

References: [1] Proteins. http://encyclopedia.farlex.com/Protein+( biochemistry). 8 January 2011. 5:15 pm. [2] Biuret Test. http://www.scumdoctor.com/nutrition/pr otein/Biuret-Test-For-Proteins.html. 8 January 2011. 5:20 pm. [3] Ninhydrin Test. http://www.chem.ucalgary.ca/courses/3 51/Carey5th/Ch27/ch27-3-3.html. 8 January 2011. 5:30 pm. [4] Xanthoproteic Test. http://mw4.merriamwebster.com/medica l/xanthoproteic%20test. 8 January 2011. 5:34 pm. [5] Millons Test. http://botanydictionary.org/millonstest.html. 8 January 2011. 5: 36 pm. [6] Hopkins-Cole Test. http://www.cerlabs.com/experiments/10 875404480.pdf. 8 January 2011. 5:40 pm.

[7] Sakaguchi Test. http://science.jrank.org/pages/41227/Sa kaguchi-test.html#ixzz2AsWo5eEV. 8 January 2011. 6:00 pm. [8] Nitroprusside Test. http://www.mondofacto.com/facts/dictio nary?nitroprusside+test. 8 January 2011. 6:04 pm. [9] Fohls Test. http://www.cerlabs.com/experiments/10 875404480.pdf. 8 January 2011. 5:41 pm.