Isolation and Characterization of Proteins

Proteins can be isolated from their native sources and purified by several methods. Egg white is known to be a rich source of dietary protein and after being subjected to salting out method its ovoglobulin content was separated from ovalbumin. The proteins were characterized using different tests and denatured through a series of denaturing conditions. The used egg white weighed 40.08g, the recovered dried ovoglobulin weighed 0.44g and the dried ovalbumin weighed 7.18g. The percentage yields were 1.10% {ovoglobulin} and 17.8% {ovalbumin} respectively. The remaining egg white was then diluted with the buffer phosphate and reagents were added to test the behavior of its protein contents. Lastly the proteins were denatured from varying conditions to alter their structure as manifested by precipitation or coagulation of the solution. Results indicate that only strong variation of acid and bases with the aid of some manipulations can influence the solution into altering the protein.

INTRODUCTION

Proteins are considered as polymers of amino acids. Proteins are biochemical compounds which consist of one or more polypeptides typically folded into a globular or fibrous form facilitating a biological function. Protein can be isolated from a native source and can be purified by several methods. Egg is a rich source of protein. The egg yolk is rich in proteins and vitamins but also has a relative high fatty acids and cholesterol content. On the other hand, the egg white contains a little more protein than the yolk but without fatty acids and cholesterol. Egg white contains many proteins but two of the main ones are albumin and globulin. Ovoglobulin can be acquired from egg white by salting out using saturated NaCl while ovalbumin can be isolated by mild acidification with acetic acid. Biuret method can be used to quantify the protein isolated and proteins when reacted with several reagents gives colored products. Ninhydrin, xanthoproteic, millon’s and Hopkins-cole test are used as general or specific test for proteins.

MATERIALS & METHODS

Isolation of Ovoglobulin and Ovalbumin from Egg white

The egg white was separated fom the yolk from one medium sized chicken egg. The yolk was discarded and the volume of the egg white was measured using a pre-weighed graduate cylinder. The weight was determined. 3.60g of NaCL powder was added for every 10ml of sample. It was stirred gently until no ovoglobulin precipitation was observed. A cheese cloth was used to filter with no undissolved salt powder included. The filtrate was not disposed. The residue was transferred to a pre-weighed filter paper & the sample was air-dried. The dried sample was weighed and the percentage yield was calculated. 2-3 drops of 0.200M acetic acid was added to the filtrate and heated to boiling point until the ovalbumin precipitates. The residue was filtered with a cheese cloth. The precipitate was transferred to a pre-weighed filter paper and was air-dried. The sample was weighed and the percentage yield was calculated.

Quantitation of protein using Biuret Method

The egg white was separated from the yolk of one medium-sized chicken egg. The yolk was discarded. The volume of the egg white was measured with a graduated cylinder. The sample was divided into to even parts with 250ml beakers for each sample. One portion was used for part C while the other was spared for part D. 2.00ml of each albumin standard was prepared into separate test tubes with concentrations of 5, 10, 15 & 25 mg/ml from standard albumin solution (50mg/ml). A blank was prepared using a 2.00ml distilled water. 2.00ml of egg white was transferred into two separate test tubes of the unknown samples. Each tube was labeled. 2,00ml of Biuret reagent & 1.00ml of 0.200M NaOH to each prepared tubes in #2. The samples were incubated for 30 minutes at room temperature. The absorbance was read at 540nm of the blank distilled using UV-Vis spectrophotometer. The absorbance of the standard was read with the highest concentration (25mg/ml). The absorbance of the samples were read and diluted until the absorbance is within that of the standard solutions. The averages of the two readings were gained. The calibration curve was prepared using MS Excel Program. The linear regression analysis was done to determine the equation of the R2 value of the line. The equation of the line was used and the concentration of the protein sample was calculated:

Equation: x=[(y-b)/m] x dilution factor x total volume of sample

Qualitative Tests for Protein

The egg white sample was diluted with equal 0.200M phosphate buffer (7.40pH) & filtered through the cheesecloth. This test was used in parts C & D. 2.00ml of egg white solution was transferred into four separate test tubes & was labeled.

• Test tube 1 – 2.00ml ninhydrin solution added, heated for 5 minutes in water bath or until color change was observed.
• Test tube 2 – 5 drops of concentrated nitric acid added, heated for 5 minutes or until color change was observed.
• Test tube 3 – 5 drops of Million’s reagent, heat for 5 minutes in water bath or until precipitate was formed.
• Test tube 4 – 5 drops of glyoxylic acid added, tube was tilted slightly & 2.ooml of concentrated sulfuric acid was added for two layers to form. The color change was observed.

Denaturation of Protein

2.00ml egg white solution was transferred to eight separate test tubes and was labeled. The following reagents were added to their respective tubes:

Test tube 1 – 0.200M HCL drop by drop until changes occurred.

Test tube 2 – 0.200M NaOH drop by drop until changes occurred.

Test tube 3 – 0.200M lead acetate drop by drop until changes occurred.

Test tube 4 – 0.200M urea drop by drop until changes occurred.

Test tube 1 – 0.200M tannic acid drop by drop until changes occurred.

Test tube 1 – 0.200M 70% ethanol drop by drop until changes occurred.

Test tube 7 – heated in water until changes occurred

Test tube 8 – agitated vigorously until changes occurred.

RESULTS AND DISCUSSIONS

The isolated egg white placed in a pre-weighed Erlenmeyer flask has a mass of 40.8g. Given the mass, the percent yield of ovoglobulin and ovalbumin was calculated. The significant results were tabulated below. The formula of percent yield is listed on Figure 1.

Ovoglobulin

Ovalbumin

Mass of the dried sample

0.44g

7.18g

Percent yield

1.10%

17.9%

The calculation of percent yield is necessary to determine the recovered proportion in terms of one hundredth. This would highlight the significant result of the experimental procedures accomplished within the scientific study if they were done qualitatively and with precision. The values above indicate that they were unavoidable errors within the process, from transferring to weighing and from filtering to drying.

Ovoglobulin and ovalbumin have certain differences. Ovoglobulin, serves as antibodies, were the one precipitated from the egg white using salting out by adding NaCl. On the other hand, ovalbumin is the one that serves as nourishment of the egg which is about 60-65% of its content. Some other proteins found on egg and their relative functions were included on the table below.

PROTEINS

%{in typical egg white}

FUNCTIONS

Ovalbumin

54%

Nourishment

Ovoglobulin

8%

Plugs defects on membrane

Ovotransferrin

12%

Binds iron

Ovomucoid

11%

Blocks digestive enzymes

Ovomucin

1.5%

Inhibits viruses, thickens the egg white

Avidin

0.06%

Binds vitamin biotin

Others

13.5%

Digestion of bacterial cell wall

Proteins showed different color reactions from the addition of several reagents. The tests involve Ninhydrin, Xanthroproteic, Sakaguchi and Hopkins Cole. The observations from these tests are shown on table 3.

The sample that was added with the ninhydrin solution showed a yellow colored reaction but it was supposed to manifest a violet color. The ninhydrin solution might be contaminated so the test showed a wrong result.

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. Millon’s 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 4.

Test Tube #

Added reagent/ Manipulation

Observations

1

HCl

precipitation of proteins

2

NaOH

precipitation of proteins

3

Lead acetate

formation of black precipitate

4

Tannic acid

protein coagulation

5

Ethanol

cloudiness or precipitation

6

Heat

coagulation of proteins

7

Agitation

foaming

Stabilizing forces or bonds were disrupted when subjected to denaturating conditions. In the heat, hydrogen bonds are broken by increased translational and vibration energy coagulation of egg white albumin in frying. By addition of strong acids and base, there were salt formations and disruption of hydrogen bonds. On some organic solvents such as ethanol, there are changes in dielectric constant and hydration of ionic groups. And on agitation, there were shearing of hydrogen bonds. The disrupted forces or bonds in de-naturating agents that were used in the experiment are shown in Table 4.1.

CONCLUSIONS

There are certain methods on how to isolate proteins. In this experiment, we used NaCl to isolate the ovalbumin content of the egg white and Acetic acid for the ovalglobulin which are both rich in proteins. There were different reactions of the egg white when it was subjected to denaturation or altering of the bonds of the proteins. This may result to disrupted stabilizing of molecules such as forming of hydrogen bonds when heating that results into the coagulation of the egg white. There were also test that show what are the contents of an egg white. It was proven that egg whites contain protein with the use of Millon’s reagent.