Isolation and Characterization of Proteins Ichiro L. Oquino, Kathleen Erika T. Orencia, Edsel F. Padolina, Erika C.

Pasion, Byran Gabriel A. Penuliar Group 6 2C Pharmacy Biochemistry Laboratory ABSTRACT

The intact protein was isolated from the samples, with casein from skimmed milk by isoelectric precipitation, albumin from skimmed milk by heat denaturation, gluten from wheat flour by difference in solubility, and myoglobin from beef heart by salt-induced precipitation. These intact proteins were characterized through the use of the different qualitative color reactions, which are the Biuret, Ninhydrin, Xanthoproteic, Millons, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohls, Test for Amide, and Pauly. The assigned protein to be isolated by the group is myoglobin from beef. The results for the qualitative color reactions of the intact protein, myoglobin, is as follows: for the Biuret test, a bluish gray solution, for Ninhydrin test, a purple solution, for Sakaguchi test, a colorless solution, for Nitroprusside test, a yellow solution, for Fohls test, a dark brown solution, for the Test for Amide, the litmus paper turned from red to blue, for Pauly test, an orange solution appeared.

INTRODUCTION
A protein is a molecule composed of polymers of amino acids joined together by peptide bonds. It can be distinguished from fats and carbohydrates by containing nitrogen. Other components include carbon, hydrogen, oxygen, sulphur, and sometimes phosphorus [1]. A protein is a linear polymer built from about 20 different amino acids. The type and the sequence of amino acids in a protein are specified by the DNA in the cell that produces them. This sequence of amino acids is essential since it determines the overall structure and function of a protein. A protein has several functions. It may serve as a structural material (e.g. keratin), as enzymes, as transporters, (e.g. hemoglobin), as antibodies, or as regulators of gene expression. A protein may be classified based on its from and main functions: it can be a globular protein like most enzymes, fibrous protein which are for structural role; and membrane proteins that serve as receptors or channels for polar or

charged molecule to pass through the cell membrane [1]. The Biuret Test is a method for detecting proteins is serum. In alkaline solution, copper sulfate ions react with the peptide bonds of protein to produce a pink to purple color, called the biuret reaction. The amount of serum protein in a sample solution is estimated by comparing its color with that of a standard solution whose protein concentration is known [2]. The Ninhydrin Test is a test for amino acids and proteins with a free NH2 group. When such an NH2 group reacts with ninhydrin, a purple-blue complex is formed [3]. Xanthoprotein is a yellow acid substance formed by the action of hot nitric acid on albuminous or protein matter and is changed to a deep orange-yellow color by the addition of ammonia. The color reaction is used to identify such proteins in the Xanthoproteic Test [4]. Millons Test is a technique used to demonstrate the presence on the amino acid tyrosine. Millons A, a mixture of sulphuric acid and mercury (II) sulphate is added to the test solution. A yellow color

indicates the presence of protein. A drop of Millons B, a sodium nitrite solution, is then added. A red color indicates a positive reaction [5]. The Hopkins-Cole Test determines the presence of the amino acid tryptophan [6]. The Sakaguchi Test is used to test for a certain amino acid and proteins. The amino acid that is detected in this test is arginine. The sample to be tested is treated with alhpa-naphthol and sodium hypochlorite. A positive result yields a reddish wine color when arginine is present [7]. The Nitroprusside Test is a test for cystinuria in which sodium cyanide and then nitroprusside are added to urine [8]. The objectives of this experiment are: (1) to isolate casein from skimmed milk by isoelectric precipitation, albumin from skimmed milk by heat denaturation, gluten from wheat flour by difference in solubitili, and myoglobin from beef heart by saltinduced precipitation; (2) to analyze chemical groups responsible for color reactions and explain the principle involved in each test; and (3) to determine the amino acid components of the proteins by thin-layer chromatography.

(NH4)2SO4 crystals ground to fine powder were then added and mixed gently until the solid dissolved. The sample was centrifuged again for 5 minutes and the supernatant was decanted off. The appearance of the purified myoglobin residue was described afterwards. 2. Qualitative Color Reactions 9 test tubes were prepared for each of the color tests. Each test tube contained an intact protein solution (0.5 g of the protein in 1 mL distilled water) and 0.5 mL of hydrolyzed sample. For the Biuret test, 20 drops of 2.5 M NaOH was added to the sampled and mixed. 203 drops of 0.1 M CuSO4 solution was then added and the test tube was shaken and the color of the solution was noted. For Ninhydrin test, 6-10 drops of 0.1% ninhydrin solution was placed into the diluted samples. The test tube was heated in a boiling water bath and the appearance of a blue-violet coloration was taken note. For Xanthoproteic test, 10 drops of conc. HNO3 was slowly added to the diluted samples, mixed, and the color of the solution was noted. Afterwards, 10 drops of conc. NaOH was slowly added, mixed, and the color of the solution was noted again. For Millons test, 5 drops of Millons reagent was added to the diluted samples and the color change was noted. For Hopkins-Cole test, 20 drops of Hopkins-Cole reagent was slowly added to the samples and mixed well. The test tube was then inclined and 20 drops of conc. H2SO4 was added slowly along the side of the test tube. The mixture was not shaken and the color at the interface was noted. For Sakaguchi test, 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution was added to the samples, mixed, and allowed to stand for 3 minutes. Afterwards, 3 drops of 2%

EXPERIMENTAL
Procedure 1. Isolation Muscle of Myoglobin from

A 6.0 g of minced beef heart (or steak) and 6 mL 70% (NH4)2SO4 solution was placed in a beaker. Afterwards, the mixture was gently stirred for 1 minute to release the myoglobin. The dark red extract was then expressed into a new beaker using a cheesecloth. The extract was then centrifuged at 13,000 x g for 5 minutes. Then, 1.5 mL of the supernatant was transferred into another empty centrifuge tube. ~0.30-0.35 g of

NaOBr was added, mixed, and the colod produced was taken note of. For Nitroprusside test, 0.5 mL of 3 M NaOH was added to 0.5 mL of the sample. Then, 0.25 mL 2% nitroprusside solution was added and the formation of a red solution was noted. For Fohls test, 5 drops 30% NaOH and 2 drops 5% (CH3COO)2Pb was added to the samples and the tube was placed in a boiling water bath. The appearance of a dark (black or brown) sediment was noted. For Test for Amides, 1 mL of 20% NaOH was added to 10 drops of the sample and the test tube was placed in a boiling water bath. Afterwards, there was a test for the evolution of gas during heating by placing a moistened red litmus paper over the mouth of the test tube. The result was then noted. For Pauly test, the diazo reagent was prepared by mixing 3-5 drops 1% sulfanilic acid with 3 drops 5% NaNO2 solution. Afterwards, 5 drops of the sample and 3-5 drops 10% Na2CO3 was added to the diazo reagent and the appearance of a red coloration was noted.

proteins also contain free amino groups on the alpha-carbon and can react with ninhydrin to produce the purple color. The Xanthoproteic test result of a pinkish solution. showed a

The Sakaguchi Test showed a result of a colorless solution. The Nitroprusside test showed a positive result of yellow solution because the cysteine group reacts with nitroprusside in alkaline solution. The Fohls test has a positive result of brown to black precipitate. The principle is about the degradation and substitution reaction to form PbS. The Test for Amides showed a positive result of yellow-orange solution and the red litmus paper turned into blue. The Pauly test showed a result of an orange solution and the red litmus paper turned to red. The positive result is a red color due to the sulphanilic acid getting diazotized in the presence of sodium nitrite and sodium carbonate with the protein sample.

RESULTS AND DISCUSSION

A. Isolation of Proteins Myoglobin is the protein that was extracted from the minced beef. The myoglobin that was extracted appeared to be color red. B. Qualitative Color Reactions The Biuret test for proteins 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 result of the biuret test for the group showed a bluish-grey color solution. The Ninhydrin Test showed a positive result of purple solution. It is because

Table 1. Results of Qualitative Color Reactions Color Reaction Intact Protein (Myoglobin) Biuret Bluish gray Ninhydrin Purple Xanthoproteic Pinkish Millons Hopkins-Cole Sakaguchi Colorless Nitroprusside Yellow Fohls Dark brown ppt. Test for Amides Red to blue litmus paper Pauly Orange Red to red litmus paper

Basic Hydroslate Clear light blue Cloudy Turbid Clear Clear Pink A bit turbid Clear with bubbles A bit turbid Clear

REFERENCES
[1] Protein http://www.biologyonline.org/dictionary/Protein Retrieved: December 29, 2012 [2] Biuret Test http://medicaldictionary.thefreedictionary.com/biuret +test Retrieved: December 29, 2012 [3] Ninhydrin Test http://www.uwplatt.edu/~sundin/351/3 51h-pro.htm Retrieved: December 9, 2012 [4] Xanthoproteic Test http://open.umich.edu/education/med/ oernetwork/pharmacology/biopharm/xa nthoproteics-test/2012 Retrieved: December 29, 2012 [5] Millons Test http://botanydictionary.org/millonstest.html Retrieved: December 29, 2012

[6] Hopkins-Cole Test http://www.reference.com/motif/scienc e/hopkins-cole-test Retrieved: December 29, 2012 [7] Sakaguchi Test http://www.reference.com/motif/scienc e/sakaguchi-test-amino-acid-proteins Retrieved: December 29, 2012 [8] Nitroprusside Test http://medicaldictionary.thefreedictionary.com/nitropr usside+test Retrieved: December 29, 2012