Isolation and Characterization of Proteins

Veronica E. Nuqui De La Salle University-Dasmarinas Dasmarinas City, Cavite

ABSTRACT Ovoglobulin and Ovalbumin were separated by isolating an egg white through salting out. The proteins were characterized based on different qualitative tests and denatured by denaturing manipulations. The isolated egg white weighed 41.4917g and the separated dried ovoglobulin weighed 2.3145g while the dried Ovalbumin weighed 2.8498. The percent yield calculated were 5.58 % and 6.87% that were low because of some errors. The other isolated egg white was diluted with phosphate buffer and used to test the behavior of proteins from the addition of various reagents and then denatured from different condition that altered the structure of proteins as confirmed by the precipitation from the solution.

INTRODUCTION Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Proteins are the basis for the major structural components of animal and human tissue. Proteins are natural polymer molecules consisting of amino acid units. The number of amino acids in proteins may range from two to several thousand. They are involved in virtually all cell functions. Each protein within the body has a specific function. Some proteins are involved in structural support, while others are involved in bodily movement, or in defense against germs. It can be isolated from their native source and then purified by different methods. These techniques depends on the differences in the molecular weight, sizes, charges, solubility and their acidbase behavior. Egg white is the common name for the clear liquid contained within an egg. It is formed from the layers of secretions of the anterior section of the hen's oviduct during the passage of the egg. It forms around either fertilized or unfertilized egg. It consists mainly of about 15% proteins dissolved in water. Its primary natural purpose is to protect the egg yolk and provide additional nutrition for the growth of the embryo, as it is rich in proteins and also of high nutritional value. MATERIALS AND METHODS The first part of the experiment was the Isolation of Ovoglobulin and Ovalbumin from Egg White. The egg white was carefully separated from the yolk. The yolk was discharged and the volume of the egg white was measured using a pre-weighed cylinder. The weight of the sample was also determined. Then, a 14.4g of NaCl powder was added It was stirred gently until there was no precipitation of ovoglobumin observed. Using a cheesecloth, it was then filtered and the undissolved salt powder was not included. The residues were transferred to a pre-weighed filter paper and the sample was air dried. The dried samples were weighed and the percent yields were calculated. 2-3 drops of 0.200M acetic acid were added to the filtrate and was heated to boiling until ovalbumin coagulated. The residue was then filtered using cheesecloth and transferred to a filter paper, air dried and weighed. The percent yields were calculated.

The remaining isolated egg white sample was used for the qualitative test and denaturation of protein. It was diluted with equal volume of phosphate buffer and then filtered with cheesecloth. For the qualitative test of protein, a 2.00ml of egg white solution was transferred to four separate test tubes and

the color change in every test was observed. For test tube 1, a 2.00 ml of ninhydrin soulution was added.. For test tube 2, five drops of concentrated nitric acid were added and for test tube 3, five drops of Millons reagent were added. For these three test tubes, they were wall heated for 5 minutes in a water bath. For the test tube 4, five drops of glyoxylic acid were added , then tilted slightly and another 2.00 ml of sulfuric acid was added to form two layers. For the denaturation of proteins, the solution was transferred to eight separate test tubes. For test tubes 1 to 5, 0.200 M HCl, NaoH, lead acetate, urea and tannic acid was dropped respectively until changes occurred. For test tube 6 to 8, 70 % ethanol was dropped, heated in water, agitated vigorously respectively until changes occurred. RESULTS AND DISCUSSIONS The isolated egg white weight was 41.4917g. From the isolation of ovoglobulin from ovalbumin in the egg white with the used of salting out, the dried sample of ovoglobulin in the filter paper was weighed and as well as the filtrate which was the ovalbumin. The percent yield calculated from the weight of the egg white minus the weight of the added salt stands as the theoretical yield and the weight of the dried ovoglobulin and ovalbumin as the actual yield. The weight of dried sample and the % yield of ovoglobulin and ovalbumin as were isolated was shown in Table 1. Table 1. Isolation of Ovoglobulin and Ovalbuin from Egg White Ovoglobulin 2.3145 5.58% Ovalbumin 2.89448 6.87%

Weight of dried sample % Yield

The percent yields were not good because of some error like filtering the samples improperly or some spill while transferring egg white or the filtrate. There were differences between the two eggwhite proteins such as ovoglobulin can be precipitated from egg white by salting out using NaCl or ammonium sulfate , while ovalbumin can be isolated by mild acidification with acetic acid. The ovalbumin is the one that serve as nourishment and the main protein found in the egg white which is approximately 60-65%. The globulin is the one that serves as antibodies in the immune system and bind to certain compounds in the body. Proteins showed different changes in color from addition of several reagents. The reagents used are: Ninhydrin, Nitric acid, Millonsreagent and Hopkins Coles reagent. Table 2. Qualitative Test for Proteins Test Tube # 1 2 3 4 Added reagent Ninhydrin Nitric acid Millons reagent Hokins Coles reagent Observations Yellow Yellow to ripe mango color Old rose complex Violet complex

The sample that was added with ninhydrin solution showed a yellow color but it supposes to show a violet color. The reason might be the ninhydrin reagent was contaminated so the test showed a wrong result. The other sample showed the write color based on the theoretical results of the tests done. Ninhydrin is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is evolved. The Xanthroproteic test is a test for proteins in which a yellow color forms on addition of conc. nitric acid and the color turns orange when made alkaline. Millons test is an analytical reagent used to detect the presence of soluble proteins. A few drops of the reagent are added to the test solution, which is then heated gently. A reddish-brown coloration or precipitate indicates the presence of tyrosine residue which occur in nearly all proteins The hopkins-cole test is used to determine the presence of the amino acid

tryptophan. Tryptophan has an indole nucleus which is responsible for the violet ring found at the junction between the two layers. It produces a purple ring at the point of contact of the two liquids. The native structures of proteins may be altered, and their biological activity be changed or destroyed by treatment that does not disrupt the primary structure. This denaturation is often done deliberately in the course of separating and purifying proteins. The denaturation of protein and the agents or condition done were shown in Table 3. Table 3. Denaturation of Protein Test Tube # 1 2 3 4 5 6 7 8 Added reagent/ Manipulation HCl NaOH Lead acetate Urea Tannic acid Ethanol Heat Agitation Observations precipitation of proteins precipitation of proteins formation of black precipitate cloudiness or precipitation protein coagulation cloudiness or precipitation coagulation of proteins foaming

Some stabilizing forces or bond were disrupted when subjected to denaturating conditions. In the heat, hydrogen bonds are broken by increased translational and vibrational energy like coagulation of egg white albumin in frying. By addition of strong acids and base, there were salt formation and disruption of hydrogen bonds. On some organic solvents such as ethanol, there were change in dielectric constant and hydration of ionic groups. And on agitation, there were shearing of hydrogen bonds. The disrupted forces or bonds in denaturating agents that were used in the experiment were shown in the table below. Table 4. Disrupted Stabilizing Forces on Denaturation Denaturating Agent/ Condition HCl NaOH Lead acetate Urea Ethanol Heat Agitation Disrupted Stabilizing Bond/Forces Salt bridges Salt bridges Disulfide bond Salt bridges Hydrogen bond Hydrogen bond Hydrogen bond

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