Name: Athina Stewart (1604349)

Lab Partner’s Name: Levonne Burke (1604299)

Date: September 29, 2017

Aim: To determine the chemical properties of amino acids, proteins and nucleoproteins

Abstract:

The solubility of phenylalanine and egg albumin were deduced by exposing each substance to
different temperatures and pHs. Protein was shown to be present in egg albumin by the positive
reaction given from the biuret test and phenylalanine was determined to be an amino acid based
off the negative reaction of the biuret test. It was also shown that various conditions could
denature proteins in egg albumin. The investigation of the nucleoprotein (conjugated protein) in
yeast, was done to show that there exists a protein and non-protein component, which would be
RNA, existed in the structure of yeast.

Introduction/Theory:

Proteins are large molecules found in every cell of the body. They are made up smaller
monomers called amino acids which are composed of C, N, O, H and small quantities of sulphur.
Amino acids are consisted of a central carbon atoms (known as the alpha carbon) bonded to a
amine group (NH2), a carboxylic group (COOH), a respective R group and a hydrogen atom.
They also display both acidic and basic properties when dissolved in an aqueous solution making
them amphoteric. Amino acids are joined together by a condensation reaction to form a protein.
The bond that is formed between amino acids are known as peptide bonds. Proteins are among
the most abundant organic molecules in the living systems and play are a variety of roles such as
transport, as digestive enzymes, structure, hormones, defense and storage. Proteins also contain
acidic and basic groups and have their own isoelectric points which is the pH at which there is no
net charge within the protein molecule.

The solubility of proteins are determined by their net ionic charge they possess. Its water
solubility is enhanced by how large the net ionic charge it has. However, when it is at its

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isoelectric point where there is no net charge (equal amount of positive and negative charges
existing), the solubility will drastically decrease and the molecule will then precipitate out of the
solution.

A protein has a specific three dimensional structure that enables it to perform its biological
activity. The three dimensional structure is dependent on hydrogen bonds, interionic bonds called
salt bridges and disulphide bonds. The destruction of the three dimensional structure is brought
upon by the breaking the sequence of the amide (peptide) bonds which is known as protein
hydrolysis and the other molecular forces of attraction present. This process is known as
denaturation.

There also exist conjugated proteins, which contains a both a protein structure and non-protein
structures. Nucleoproteins is an example of conjugated protein, where a nucleic acid (RNA and
DNA) is bonded to a protein. The non-protein part of a conjugated protein can be broken by acid
hydrolysis. The parts that RNA is a phosphate, a sugar (ribose) and a organic base (a purine).

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Pre-Lab Questions:

The isoelectric points of four amino acids: glycine, lysine, glutamic acid and phenylalanine as
well as egg albumin were found. Additionally it was stated whether or not each is non-polar or
polar: neutral, basic or acidic.

Amino Acid or Protein Isoelectric Point / pI Polar/Non-Polar

Phenylalanine 5.48 Neutral
Lysine 9.59 Basic
Glycine 5.97 Neutral
Glutamic Acid 3.22 Acidic
Egg Albumin 4.8 acidic

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Apparatus/Materials: 0.2% phenylalanine solution, yeast, physiologic saline solution, 10%
sodium hydroxide, 15% sodium hydroxide, dilute copper (II) sulphate, 0.2% hydrochloric acid,
0.5% sodium carbonate, egg albumin solution, tannic acid, mercuric chloride, ethanol, 3M
sulphuric acid, Bial’s Orcinol reagent, concentrated ammoniu hydroxide, silver nitrate, nitric
acid, ammonium molybdate, distilled water, hot plate, 400 ml beaker, test tubes, test tube holder,
tongs.

Procedure:

Four groups of tests were carried out to determine the characteristics of phenylalanine and egg
albumin, as well as a nucleoprotein in yeast.

The first test was to determine the solubility of phenylalanine and egg albumin. 2mL of 0.2%
phenylalanine solution was added to six test tubes and to each, 2mL of cold water, hot water,
physiologic saline solution, 10% sodium hydroxide solution, 0.2% hydrochloric acid and sodium
carbonate were added respectively. It was then observed whether or not phenylalanine stayed
dissolved or precipitated and this test was repeated with egg albumin solution. The results were
recorded.

Secondly, the denaturation of egg albumin was tested. This was done by adding 2mL of egg
albumin into a beaker and four test tubes. The beaker was heated on a hot plate and 2mL of
alcohol, tannic acid and mercuric acid were added to the remaining three respectively. The fourth
was vigorously shaken for 2 minutes. Observations and results were recorded.

Thirdly, phenylalanine and egg albumin solution were tested for the presence of proteins. 2mL of
egg albumin mixture was added to a test tube. 2mL of 15% sodium hydroxide was then added
and mixed. 5 drops of dilute copper (II) sulphate was then added to the tube. Observations and
colour changes were recorded. This test was then repeated with phenylalanine.

Lastly, protein, ribose, phosphate and purine were then tested for in yeast. A small amount of
yeast was mixed with 2mL of water and shaken vigorously. Protein was then tested for using the
Biuret test (the third test). Then an RNA solution was prepared using 0.1g of yeast, 3ml of
distilled water and 3mL of 3M sulphuric acid. It was then heated for 20 minutes at 80°C. 1mL of

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the solution was then added to three test tubes. First ribose was tested for by adding 2mL of
water and 3mL of Bial’s Orcinol reagent to the first test tube. It was then mixed and heated in a
boing water bath for 10 minutes. Secondly, purine was tested for by adding concentrated
ammonium hydroxide solution until it became basic. 5 drops of silver nitrate was then added to
the solution. Lastly, phosphate was tested for by again making the solution basic with
concentrated ammonium hydroxide and then it was turned slightly acidic with nitric acid. 2mL of
ammonium molybdate was then added and the solution warmed. All observations and results
were recorded.

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Results:

TABLE SHOWING SOLUBILITY OF COMPOUNDS

Phenylalanine Egg Albumin

Cold Water No precipitate
Hot Water No precipitate
Saline Solution Precipitate Formed
Sodium Hydroxide No precipitate
(pH>14)
Hydrochloric acid (pH No precipitate
1-2)
Sodium carbonate Precipitate Formed
(pH 9-10)

TABLE SHOWING DENATURATION OF COMPOUNDS

Observations
Heat Precipitation seen / Denaturation took place
Alcohol No change / No denaturation
Tannic Acid No change / No denaturation
Mercuric Chloride Precipitation seen / Denaturation took place
Shaking Foam seen / Denaturation took place

TABLE SHOWING TEST FOR PROTEIN

Substance Observations
Egg Albumin Purple solution / Protein present
Phenylalanine Solution remained blue / No protein present

TABLE SHOWING THE RESULTS OBTAINED FROM THE INVESTIGATION OF THE NUCLEOPROTEIN

Substance Observations
Protein Purple solution
Ribose Green solution
Purine White precipitate
Phosphate Yellow precipitate

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Discussion:

For this experiment, the solubility of phenylalanine and egg albumen was investigated using
different substances and at different temperatures (hot and cold water); the effect of different
chemical compounds on the denaturation of egg albumen; the identification of a protein
molecule using the biuret test and the investigation of the protein and non-protein part of a
nucleoprotein were all tested for.

The denaturation of a protein deals with the disruption of the bonds that holds the three
dimensional structure in place. In the experiment, this denaturation process was investigated
using proteins existing in egg albumin. When the egg albumin was subjected to heat, it was
observed that the egg albumin started to precipitate given an indication of denaturation.
Therefore, high temperatures denatures proteins, this is due because it aggravates the interionic
bonds, hydrogen bonds etc. by adding more energy to them eventually breaking them. Secondly,
when alcohol is added to the egg albumin, it disrupts the hydrogen bonds within the three
dimensional structure causing denaturation which is observed when the precipitation occurred.
When mercuric acid was added, the heavy metal ions present (mercury ions) interacts with the
carboxylate anions of the acidic or sulphide bonds disrupting them causing the protein to
precipitate signifying denaturation. When the egg albumin was shook vigorously, the evolution
of foam was seen indicating denaturation. This vigorous motion disrupted the bonds that held the
protein structure together.

In the identification of egg albumin and phenylalanine has proteins, the biuret test was used. The
Cu2+ ions from the Biuret’s solution forms a purple chelate complex with the peptide bonds
within the protein. Based on the results obtained, egg albumin contained protein molecules while
phenylalanine was absent of it.

In the investigation of the nucleoprotein in yeast gave positive results for both the protein and
non-protein component. The protein component was tested for using biuret’s reagent which gave
a colour change to violent indicating that a protein is indeed present. The non-protein parts
(RNA) was tested for using specific tests. Firstly, when the Bial’s Oricinol reagent and water
was mixed with the RNA solution and heated, it gave a green colour which indicated a hexose
sugar (ribose) was present. This colour change was seen due the dehydration of hexose forming

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5-hydroxyl-fufural which later reacts with oricinol and Fe3+. Secondly, when the RNA solution
was made basic by the addition of ammonium hydroxide and then a few drop of sliver nitrate
was added to the mixture, it precipitated the purine base within the RNA structure. This was
observed because the hydrolysis of bonds between the purine bases and ribose results in the
release of purine bases caused by ammonium hydroxide. Ag+ then precipitates from the sliver
nitrate. Finally, when the RNA solution was made basic with concentrated ammonium hydroxide
then slight acidic with nitric acid then again mixed with ammonium molybdate and warmed,
yield a yellow precipitate indicating the presence of phosphate. This was observed due to the
hydrolysis of pyrophosphate to phosphate.

Errors:

Questions:

A. Illustrate by drawing the 3 Glycine structures with charges of +1, 0, -1 and explain how
the pH at which the pI is found is calculated from the pKa of Glycine. Then explain the
others by comparison.
Solution

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 The pI is the pH at which the amino acid has no overall charge. The range of pHs were
the overall charge of the majority of the molecules of zero.for glycine, this happens
between the pkas 2.2 to 9.6. the pKa is the measure of the acidity of a solution. The
average pKa is then taken to be 5.9. for amino acids with acidic side chains, the
average of the first two pKas are usually used for amino acids with basic side chains,
the higher two pKas are used.

B. Explain the solubility properties based on pH and isoelectric points.

Solution
The pH can affect solubility in two ways. Firstly, an insoluble compound could be
dissolved in a solvent possessing similar ion. For example, calcium hydroxide could be
dissolved by NaOH and the pH of the solution will increase, therefore shifting the
equilibrium position of the dissociation of Ca(OH)2 to the left. Additionally,

C. Describe protein folded structure in terms of bonds and forces of attraction to explain
your results.
Solution
Proteins are formed when amino acids are bonded together by peptide bonds. This
occurs when the alpha amide of the amino acid is bonded to a carboxylic group in
another amino acid via condensation reactions. Multiple amino acids joined together
give a polypeptide. When forming a protein, polypeptide are made longer by adding
‘C’ and ‘N’ terminals. In the primary structure, the sequence of amino acids that make
up the protein is displayed. In the secondary structure, the peptide bond is stabilized
by hydrogen bonds between the N-H and C=O groups causing it to begin to fold. In the
tertiary structure however, the functional groups in the side chains begin to interact
with each other via different forces of attraction forming a three dimensional

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 structure. These forces of attraction include: ionic bonds, hydrogen bonds and
disulfide bonds. In a quaternary structure, involves more than one polypeptide chains.

D. Draw the complex. Explain the origin of the name as a test for the compound biuret.
Solution

The Biuret reagent does not contain biuret, but was named so because it gives a
positive reaction to the peptide bonds in biuret.

E. Discuss what yeast is and what nuclei acids, to explain your results. Draw the structure
of a nucleotide of RNA using Adenine as the purine.
Solution
Yeast is a eukaryotic, single celled fungi. Yeast is commonly found throughout native
and within the human body. Nucleic acids are composed of nucleotide monomers
linked together and they allow the genetic transfer of information within organisms.

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References:

www.harpercollege.edu

https://prezi.com/m/vfjrplptge8b/test-for-carbohydrates-bial-and-iodine/

www.jbc.org/content/176/2/703.full.pdf

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