Isolation And Characterization Of Proteins Biology Essay

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INTRODUCTION

Proteins are macromolecules. They are constructed from one or more unbranched chains of amino acids; that is, they are polymers. (2) The protein in eggs has a biological quality greater than any other natural food. In fact, some manufacturers of protein powders (such as albumin) often base their products on egg protein because of its protein quality. Egg protein contains all the essential amino acids in the exact proportions required by the body for optimum growth and maintenance of lean, metabolically active muscle tissue. (3) White is the common name for the clear liquid (also called the albumen or the glair/glaire) contained within an egg. In chickens it is formed from the layers of secretions of the anterior section of the hen's oviduct during the passage of the egg. (4) It forms around either fertilized or unfertilized yolks. The primary natural purpose of egg white is to protect the yolk and provide additional nutrition for the growth of the embryo.

MATERIALS AND METHODS

First, the ovuglobulin and ovalbumin were isolated from the egg white. From a chicken egg the egg white was carefully separated from the egg yolk, and then the volume of the egg white was weighed using the analytical balance. The isolated egg was divided into two containers, the first container is for the isolation process and the other one is for the characterization process of egg white proteins. The 2nd division of the isolated egg was equally distributed and each contains 10ml egg white. 3.06g NaCl powder was added to all test tubes; it was stirred gently until change is observed. After that the other division was filtered with cheesecloth to separate the produce filtrate. The remains were placed in a filter paper to cool and dry. Dried sample was weighed and the percentage yield was calculated for ovoglobulin. The filtrate was placed on a beaker having 2-3 drops of 0.200 acetic acid; it was heat until ovalbumin precipitates. The produce residue was placed again in a filter paper for it to cool and dry, and then the dried sample was weighed and percentage yield was calculated this time for ovalbumin. The leftover isolated egg whites were used in the denaturing and qualitative test of proteins. The egg white was diluted with phosphate buffer and filtered using the cheesecloth. For the qualitative test of proteins, 1.00ml egg white was transferred to four separate test tubes and the change of color was observed.

1 2.00ml of ninhydrin solution; heat gently in water bath

2 5 drops of conc. nitric acid (Xanthroproteic test); heat gently in water bath

3 5 drops of α-naphthol reagent,2.00ml 0.200M NaOH & 5drops of brominewater (Sakaguchi test

4 5 drops of glyoxylic acid (Hopkins-Cole test), 2.00ml conc. sulfuric acid

The egg that was diluted was used for the denaturation of protein, 1.00ml was transferred to eight separate test tubes and labeled accordingly. The reagents and procedure that was added and done are listed below. All reagents were added drop by drop to observe accurate change.

1 0.200 M HCl

2 0.200 M NaOH

3 0.200 M lead acetate

4 0.200 M tannic acid

5 70% ethanol

6 Heat in water bath

7 Agitate vigorously

There are only 7 procedures than because sample in urea is not available.

RESULTS AND DISCUSSION

The total weight of the isolated egg white is 25.864. from the isolation of ovoglobulin and ovalbumin, the dried sample of ovoglobulin was weighed 4.564 and the ovalbumin was weighed 3.479. the percentage yield was calculated from the weight of the egg white minus the weight of the additional salt which is 10.8397g as stated, and the actual yield are the the value of the dried ovoglobulin and ovalbumin.

Ovoglobulin

Ovalbumin

Weight of dried sample

4.564

3.479

% yield

30.457 %

23.217 %

The calculations for the percent yield were shown below :

Theoretical value = 37.6314g - 10.8397 = 26.7917g

Ovoglobulin

x 100 = 17.04

Ovalbumin

x 100 = 12.99

The yields calculated may seem low due to some errors during the experiment like, some spills were done and maybe the filtration was not very accurate. Egg white consists primarily of about 90% water into which is dissolved 10% proteins (including albumins, mucoproteins, and globulins). Unlike the yolk, which is high in lipids (fats), egg white contains almost no fat, and the carbohydrate content is less than 1%. Egg white has many uses in food, and many others, including the preparation of vaccines such as those for influenza (5)

Qualitative Test for Proteins

Test Tube

1

Added Reagent

Color Observations

2

Ninhydrin solution

Yellow

3

Conc. nitric acid

Yellow to Orange

α-naphthol reagent NaOH &Bromine watered

Red

4

Glyoxylic acid conc. sulfuric acid (exothermic)

Violet

Some of the results are wrong like the ninhydrin solution, after the reagent was added it showed a yellow color change when supposedly it should have possess a violet color, this kind of result is maybe due to contaminations of the solutions. But nevertheless all aother test showed correct data.

Denaturation of Protein

Test tube

Denaturing agent

Observations

1

0.200M HCl

Coagulate

2

0.200M NaOH

White precipitate

3

0.200M lead acetate

White homogenize

4

0.200M tannic acid

Brown with residues

5

70% ethanol

Coagulate

6

Heat in water bath

Coagulate

7

Agiate vigorously

bubbles

This table is the representation of the denaturation process of proteins. Some results were considered correct and some are in great discussion whether to be considered or not, these results are all from the product after heating the egg white sample with some denaturing reagents. On some organic solvents such as ethanol, therewere change in dielectric constant and hydration of ionic groups.

Disrupted Stabilizing Forces on Denaturation

Denaturing agent

Disrupted stabilizing force

0.200M HCl

Salt brigdes

0.200M NaOH

Salt brigdes

0.200M lead acetate

Disulfide bonds

0.200M tannic acid

Salt brigdes

70% ethanol

Hydrogen bonding

Heat in water bath

Hydrogen bonding

Agiate vigorously

Hydrogen bonding

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