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, Millon¶s, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohl¶s, 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 Millon¶s Test was done by adding 5 drops of Millon¶s 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 Fohl¶s 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 Millon¶s Test, violet ring at the interface for Hopkins-Cole Test, red-orange solution for Sakaguchi Test, yellow solution for Nitroprusside Test, black ppt. for Fohl¶s 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 . 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 . 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
Fohl¶s test is used to know if sulfur-containing amino acids are present .0 g minced beef was placed with 6 ml 70 % (NH4)2SO4 solution in a small beaker. The supernatant was decanted off and then the appearance of the purified myoglobin residue was described. Sakaguchi. The insoluble material was the crude gluten. Ninhydrin. Xanthoproteic. i. Millon¶s. Sakaguchi test is a test for guanidines. In Biuret Test. For Ninhydrin test. The mixture was gently stirred for 1 minute to release the myoglobin. Nitroprusside. A 1. gluten from wheat flour by difference in solubility.30-0.5 g of intact protein in 1 mL distilled water. Isolation of Myoglobin from muscle A 6.1 M CuSO4 solution were added to the samples.e arginine and peptides that contain it . Hopkins-Cole. The samples were centrifuged again for at least 5 minutes. The washings was tested with I2 solution until a negative results is obtained. 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 . There were 9 test tubes prepared for each of the test reaction. 6-10 drops of 0. A ~0. 20 drops of 2. they were shaken and then observed for color changes. The casein residue was dried then its weight % was calculated.0 grams and 50.
Isolation of Gluten from Wheat Flour Water was added to 1 cup of wheat flour to make thick dough. The extract was centrifuged at 13 000 x g for 15 minutes. Test for amides is used to detect R-groups of asparagines and glutamine. Ninhydrin test is typical for amino acids .bonds . A 10% acetic acid was added dropwise when the mixture has reached 400 C and then stirred gently after every 5 drops. The mixture was then heated in a temperature of about 400 C. Millon¶s test is used to demonstrate the presence of the amino acid tyrosine .0 ml of water was placed into a 100-ml beaker. The congealed casein was filtered by using gravity filtration and then the decantate was set aside for the isolation of albumin.1 % Ninhydrin solution was added to the
. Each test tube consisted of 0. The objectives of this experiment are (1) to isolate casein from skimmed milk by isoelectric precipitation. The dark-red extract was expressed into a new beaker using cheesecloth. and myoglobin from beef by salt-induced precipitation. Experimental: Isolation of Casein from Skimmed Milk Powdered non-fat milk with a weight of 2. Acetic acid was added continuously until the pH reaches 4.5 ml of supernatant was transferred into another empty centrifuge tube. Fohl¶s and Amide. HopkinsCole test is specific for tryptophan group . Qualitative Color Reactions of the Intact Proteins The intact proteins were tested with different characterization tests namely: Biuret. and (2) to analyze chemical groups responsible for color reactions and explain the principle involved in each test.35 of (NH4)2SO4 crystals ground was added to fine powder then mixed gently until the solid dissolves. Afterwards.5 M NaOH and 2-3 drops of 0. Nitroprusside test is a test for cystinuria.6. The dough was wrapped in the cheesecloth then placed under running water until all starch is removed.
6. the insoluble material is the gluten. In Millon¶s test. gluten.02 % -naphthol solution was added to the samples then mixed and was let stand for 3 minutes. 5 drops of 30 % NaOH and 2 drops of 5% (CH3COO)2Pb were added to the samples then placed in a water bath. Results obtained from the tests
Color Reaction Biuret Ninhydrin Xanthoproteic Millon¶s Hopkins-Cole Sakaguchi Nitroprusside Fohl¶s Test for Amide
Intact Protein (casein. In Fohl¶s Test.25 ml 2 % nitroprusside solution was added. 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. Appearance of black precipitate was noted. 3 drops of 10% NaOBr was added. Color changes after each addition were observed. Lastly. Qualitative Color Reactions of the Intact Proteins
Table 1. and they are least soluble at their pI
because there are no net electrostatic repulsions between protein molecules. In Sakaguchi test. yellow-orange solution
The Biuret Test for proteins positively identifies the presence of proteins in solution with violet color. In Nitroprusside Test. 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.5 ml of 3M NaOH and 0. This was done by washing the dough with water. In Xanthroproteic test. This removes the starch. The appearance of a blueviolet coloration was taken note of. 5 drops of Millon¶s reagent was added to the diluted sample with the color taken note of. concentrated nitric acid and concentrated sodium hydroxide. Formation of a red solution was noted. a pH at which they have no net charge. myoglobin) violet solution blue-violet solution orange solution flesh ppt. 20 drops of Hopkins-Cole reagent was slowly added to the sample and mixed well. 0. (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. 10 drops each of 10% NaOH and 0. violet ring at the interface red-orange solution yellow solution black ppt red to blue litmus paper. (b) gluten from wheat flour Gluten was isolated from the wheat flour by solubility differences. an acid is used to adjust the pH to 4. pI. The color at the interface was noted. It was then mixed and color change was noted. were added slowly and then mixed. Results and Discussions: Isolation of Proteins (a) casein from skimmed milk Casein was isolated from the skimmed milk by isoelectric precipitation. So to isolate casein at its isoelectric pH.
. For Hopkins-Cole test. The test tube was inclined to add slowly 20 drops of concentrated H2SO4. 10 drops each. Afterwards.diluted sample and heated in a boiling water bath. Biuret reacts with copper (II) ions in a basic solution to form a violet complex. All proteins have an isoelectric point. Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract.
The principle is about the
degradation and substitution reaction to form PbS. The Fohl¶s Test (Lead (II) acetate Test) has a (+) result of brown to black precipitate.org/millonstest. The Sakaguchi Test has a (+) result of red to red-orange color.farlex.chem.html.  Biuret Test.com/experiments/10 875404480. http://www.  Xanthoproteic Test.com/nutrition/pr otein/Biuret-Test-For-Proteins. 8 January 2011. 8 January 2011. The Millon¶s Test showed a positive result of flesh precipitate. So proteins with tyrosine will show a positive result.  Hopkins-Cole Test.ucalgary. 8 January 2011. 5:30 pm. The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet ring product. http://botanydictionary.  Ninhydrin Test.com/Protein+( biochemistry).  Millon¶s Test. http://encyclopedia. 5:34 pm. 5:40 pm. The nitrated tyrosine complexes mercury (I) and mercury (II) ions into the solution to form old rose/flesh to red precipitate. 8 January 2011.pdf. The principle around this is the nitration of aromatic rings via SEAR. http://mw4. In this test. It is because proteins also contain free amino groups on the alpha-carbon and can react with ninhydrin to produce the blue-violet color. Colorimetric Results
References:  Proteins. 5:15 pm.
. http://www. The Xanthoproteic Test showed a positive result of orange solution. http://www. 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.merriamwebster. the phenol group of the tyrosine was nitrated by nitric acid.html.com/medica l/xanthoproteic%20test.ca/courses/3 51/Carey5th/Ch27/ch27-3-3. The Hopkins-Cole Test showed a positive result of violet ring at the interface.cerlabs. 5:20 pm. 8 January 2011.html. 5: 36 pm. 8 January 2011.scumdoctor.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. The Test for Amide showed a positive result of yellow-orange solution and the red litmus paper turned into blue.
http://www. 8 January 2011.mondofacto.com/experiments/10 875404480.jrank.  Fohl¶s Test.pdf.  Nitroprusside Test. http://www. Sakaguchi Test. 5:41 pm.
.html#ixzz2AsWo5eEV. http://science.com/facts/dictio nary?nitroprusside+test.cerlabs. 8 January 2011.org/pages/41227/Sa kaguchi-test. 6:04 pm. 8 January 2011. 6:00 pm.