Prac write up

1. FACULTY: MEDICINE AND HEALTH SCIENCES
PROGRAMME: BMS
COURSE: BMS BIOCHEMISTRY
PRACTICAL: THREE (3)
REGISTRATION: R235053A
NAME: ANOTIDA
SURNAME: KACHEPA
TITLE: PROPERTIES OF PROTEINS
DUE DATE: 31/10/2023

2. INTRODUCTION
Proteins are complex organic macromolecules composed of amino acids linked together by
peptide bonds. They are essential building blocks of life and play numerous critical roles in cells
and organisms (Voet.D, 2015).
Proteins are involved in a wide range of biological functions, including:
Structural Support: Proteins provide structural support and stability to cells and tissues. They
form the structural framework of various components, such as collagen in connective tissues and
keratin in hair and nails.
Enzymatic Catalysis: Many proteins serve as enzymes, which are catalysts that facilitate
chemical reactions within cells. Enzymes accelerate the rate of biochemical reactions, allowing
essential processes such as digestion, metabolism, and DNA replication to occur.
Transport and Storage: Some proteins, such as hemoglobin, transport oxygen in the blood, while
others facilitate the movement of molecules across cell membranes. Proteins also act as storage
molecules for important substances, such as iron (ferritin) and amino acids (albumin).
Immune Response: Antibodies, a type of protein, are crucial components of the immune system
and help recognize and neutralize foreign substances (antigens) such as bacteria or viruses.
Cell Signaling: Proteins participate in cell signaling pathways by transmitting and receiving
signals within and between cells. Examples include receptors on cell surfaces that detect
hormones or neurotransmitters and initiate specific cellular responses.
Muscle Contraction: Proteins such as actin and myosin are responsible for muscle contraction,
enabling movement and locomotion.
The primary structure of a protein is the specific sequence of amino acids linked together in a
chain. The sequence is determined by the genetic code encoded in an organism's DNA. The
sequence of amino acids influences the folding and three-dimensional structure of the protein,
which is crucial for its function.
Proteins can have different levels of structural organization:
Primary Structure: The linear sequence of amino acids in a protein chain.
Secondary Structure: Localized folding patterns formed by hydrogen bonding, resulting in
structures like alpha helices and beta sheets.
Tertiary Structure: The overall three-dimensional folding of a protein, including interactions
between amino acid side chains.

3. Quaternary Structure: Some proteins consist of multiple subunits that come together to form a
functional protein complex.
The structure of a protein is intimately linked to its function. Changes or disruptions in protein
structure, such as denaturation, can impact its biological activity and may lead to dysfunction or
loss of function.
Important classes of dynamic proteins are the enzymes. They catalyze chemical reactions,
converting a substrate to a product at the enzyme's active site. Almost all of the thousands of
chemical reactions that occur in living organisms require a specific enzyme catalyst to ensure
that reactions occur at a rate compatible with life (Brody. L, 2023 October 27).
Protein structures have evolved to function in particular cellular environments, therefore they
work best at certain optimum ranges. Conditions different from those in the cell can result in
protein structural changes, large and small. A loss of three-dimensional structure sufficient to
cause loss of function is called denaturation. Most proteins can be denatured by heat, which
affects the weak interactions in a protein (primarily hydrogen bonds) in a complex manner.
Denaturation is a process in which proteins or nucleic acids lose the quaternary structure,
tertiary structure and secondary structure which is present in their native state, by application of
some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an
organic solvent (e.g., alcohol or chloroform), radiation or heat. If proteins in a living cell are
denatured, this results in disruption of cell activity and possibly cell death. Denatured proteins
can exhibit a wide range of characteristics, from loss of solubility to communal aggregation.
When a protein is denatured, secondary and tertiary structures are altered but the peptide bonds
of the primary structure between the amino acids are left intact.The external stresses interfere
with the ionic,disulphide,hydrogen bonds and the weak van der Waals forces that stabilize the
protein structure thus resulting in denaturation of the proteins (Nelson and Cox,2005). Therefore
the objectives of this practical were to demonstrate some properties of proteins, and their activity
in different conditions.
METHOD
0.5% albumin was used as the protein sample in this experiment. Various tests including the
Biuret test, Xanthoproteic reaction, Use of sulphosalicyclic acid, Esbach’s reagent, exposure to
heavy metal cations and anions, and exposure to extremes of Ph, were performed on this protein.
The tests are described in detail in Table 1.

4. RESULTS
Table 1: Test procedures performed on albumin, observations and conclusions
Test procedure Observation Conclusion
2 A: 5 drops of 5% NaOH
were added to 2ml
albumin, It was mixed
0.5ml freshly prepared 2%
sodium nitroprusside
solution was added.
light yellow solution formed Protein is denatured
B: 5 drops of 5% NaOH
were added to 2ml
albumin it was mixed and
boiled for 10 seconds
0.5ml freshly prepared 2%
sodium nitroprusside
solution was added
deep yellow solution formed Protein is not denatured
3 A: 5 drops of plasma were
added to 2ml of 95%
ethanol. 10ml of water
were added then mixed
off-white dense precipitate
noted on addition of plasma.
Ppt dissolved upon addition of
water.
Protein denatured thus it
precipitated
B: 5 drops of plasma were
added to 2ml of 95%
ethanol. The tube was
allowed to stand for 45
minutes and 10 ml of
water were added.
White sediment ppt and pale
yellow supernatant. Ppt
dissolved upon addition of
water.
Protein denatured thus it
precipitated
Test procedure Observation Conclusion
4 A: 3 drops of 2% lead
acetate were added to 2ml
albumin.
light, white solid, soluble in
excess
Protein denatured thus it
precipitated
B: drops of 2% silver
nitrate were added to 2ml
albumin.
most dense white solid,
insoluble in excess
Protein denatured thus it
precipitated
C: 3 drops 4% mercuric
chloride were added to
2ml albumin.
least dense white solid Protein denatured thus it
precipitated
5a A few drops of 20%
sulphosalicyclic acid was
added to 5ml albumin.
white ppt formed Protein present
b 3ml of albumin was
treated with an equal
volume of Esbach’s
reagent (solution of picric
thick yellow ppt Protein present

5. and citric acids in water).
c 0.5ml of 10%
trichloracetic acid was
added to 3ml albumin.
white ppt Protein present
6a 2ml saturated ammonium
sulphate was added to 2ml
plasma, it was mixed. 4ml
of water was mixed.
white ppt which dissolved
upon addition of water
Protein precipitated from
solution
b Add 5ml saturated
ammonium sulphate to
5ml plasma. Mix and filter
the ppt till clear filtrate is
obtained.
white ppt upon addition of
ammonium chloride.
clear filtrate obtained upon
filtration.
Protein is precipitated from
solution
c Add 1 drop of 1% acetic
acid and boil for 10
seconds
white coagulum formed Albumin present
Add a small spoonful of
(NH4)2SO4 crystals and
shake. Filter and test for
protein.
no coagulum formed Albumin/Protein absent
7 5 drops of 1% CuSO4
solution followed by 4ml
5M NaOH to 2ml
albumin were added
(Biuret test)
Colour change from blue to
purple Protein present
8 A few drops of conc. nitric
acid to 2ml of 0.5%
albumin. It was heated
until any ppt which was
formed on addition of acid
dissolved. It was Cool and
6M NaOH was added in
excess.
white ppt formed, dissolves in
excess
yellow colour of solution
turns to orange
Protein with amino acids
carrying aromatic groups
present, especially tyrosine

6. DISCUSSION
. In experiment 2. The addition of 0.5ml of a freshly prepared 2% sodium nitroprusside solution
subsequently leads to the formation of a light yellow precipitate in tube A. This reaction occurs
due to the interaction between the sodium nitroprusside and the modified albumin. The light
yellow precipitate is formed when the nitroprusside-albumin complex aggregates or precipitates
from the solution. Boiling the solution as was done in tube B causes an increase in temperature,
which can lead to the breakdown of the nitroprusside-albumin complex. The high temperatures
promote the decomposition of the complex resulting in denaturation and the release of the
nitroprusside ligand from the albumin. As the nitroprusside ligand is released, it undergoes
further chemical reactions, leading to the formation of new compounds. These compounds are
responsible for the deepening of the yellow color and the formation of a deep yellow precipitate.
In experiment 3 when ethanol comes into contact with plasma, it causes changes in the
conformation and stability of the proteins present, leading to denaturation. Denatured proteins
tend to lose their solubility and may form aggregates or precipitates, appearing as a white
cloudiness in the solution as was seen in tube A.
On standing, the white precipitate may undergo further changes, including a color shift to a pale
yellow as was seen in tube B. This change in color is typically attributed to the oxidation of
certain components in the plasma, such as bilirubin. Bilirubin is a yellow pigment derived from
the breakdown of red blood cells, and its oxidation can lead to the formation of yellow-colored
compounds. The pale yellow color may also be influenced by the presence of other substances in
the plasma that can undergo oxidative or chemical reactions over time. These reactions can result
in the production of compounds with yellow hues, contributing to the observed color change.
In experiment 4 when drops of lead acetate were added to tube A albumin, a chemical reaction
occurred between the lead ions (Pb² ) from lead acetate and the amino acid residues present in
⁺
albumin. This reaction can lead to several outcomes such as formation of a Lead-Albumin
Complex which lead to denaturation and formation of a cloudy or insoluble precipitate. In the
case of excess lead acetate, the concentration of lead ions is relatively high compared to the
available binding sites on albumin. This enables multiple lead ions to bind to different amino
acid residues on the albumin molecule, forming a complex that remains soluble in the solution
therefore not precipitating. In tube B formation of dense precipitate is due to the reaction
between silver ions (Ag ) from silver nitrate and the amino acid residues present in albumin.
⁺
Silver ions have a strong affinity for sulfur-containing amino acids, particularly cysteine
residues, present in proteins like albumin. The reaction between silver ions and cysteine residues
leads to the formation of silver-cysteine complexes, which are relatively insoluble in water. The
interaction between silver ions and albumin can result in the cross-linking or aggregation of
albumin molecules, leading to the precipitation of albumin. The formation of these insoluble
complexes causes the albumin to separate from the solution, forming a dense precipitate.
In experiment 5 tube A sulphosalicyclic acid was used. It is a white or faintly pink crystalline
substance that is highly water soluble and is used as a reagent in tests for albumin and as an
intermediate compound in the manufacture of dyes and surfactants. This test is used as a test for
proteins in urine (clinical application). Then in tube B and C white and yellow precipitates were
formed due to the formation of complexes indicating the presence of proteins.

7. Experiment 6 was salting out with ammonium sulphate. Ammonium sulfate precipitation is a
method used to purify proteins by altering their solubility. It is a specific case of a more general
technique known as out. Ammonium is commonly used as its solubility is so high that salt
solutions with high ionic strength are allowed. The solubility of proteins varies according to the
ionic strength of the solution, and hence according to the salt concentration. Two distinct effects
are observed: at low salt concentrations, the solubility of the protein increases with increasing
salt concentration (i.e. increasing ionic strength), an effect termed salting in. As the salt
concentration (ionic strength) is increased further, the solubility of the protein begins to decrease.
At sufficiently high ionic strength, the protein will be almost completely precipitated from the
solution (salting out).Since proteins differ markedly in their solubilities at high ionic strength;
salting-out is a very useful procedure to assist in the purification of a given protein. The
commonly used salt is ammonium sulfate, as it is very water soluble, forms two ions high in the
Hofmeister series, and has no adverse effects upon enzyme activity. It is generally used as a
saturated aqueous solution which is diluted to the required concentration, expressed as a
percentage concentration of the saturated solution (a 100% solution).
In experiment 7, a biuret test was performed on albumin. The biuret test is a chemical test used
for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms
violet-colored coordination complexes in an alkaline solution. This color change is dependent on
the number of peptide bonds in the solution, so the more protein, the more intense the
change. The NaOH is added in order to raise the pH of the solution to alkaline levels whereas the
crucial component of this test is the copper II ion (Cu2+) from the CuSO4 ( Ruiz GA et al ,
20219).
Experiment 8 was the xanthoproteic reaction. This is a method that can be used to determine the
amount of protein soluble in a solution, using concentrated nitric acid. The test gives a positive
result in those proteins with amino acids carrying aromatic groups, especially in the presence of
tyrosine. If the test is positive the proof is neutralized with an alkali, turning dark yellow. The
yellow colour is due to xanthoproteic acid which is formed due to nitration of certain amino
acids, most common examples being tyrosine and tryptophan. This chemical reaction is a
qualitative test, determining the presence or absence of proteins. 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 colour by the addition of ammonia.

8. Answers to questions
1) Proteins are polymers of amino acids. Each protein is made up of fifty or more amino
acid subunits. The sequence of amino acids in the polypeptide chain determines how the
protein is folded and how it attains its 3-dimensional structure. Examples of proteins are
actin,insulin,maltase and collagenase.
2) Denaturation is a process in which proteins or nucleic acids lose the quaternary structure,
tertiary structure and secondary structure which is present in their native state, by
application of some external stress or compound such as a strong acid or base, a
concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or
heat. If proteins in a living cell are denatured, this results in disruption of cell activity and
possibly cell death. Denatured proteins can exhibit a wide range of characteristics, from
loss of solubility to communal aggregation
CONCLUSION
Proteins have different properties which are due to their differences in the side chain groups and
also due to the 3-dimensional structure of the proteins. The functionality of a protein is affected
by salts,pH,heavy metals and also extremes in temperature which disrupt their 3-dimensional
structure. These denaturation agents should be maintained within optimum ranges if the proteins
are to perform their functions properly.

9. REFERENCES
Brody.L (2023 October 27) Protein
https://www.genome.gov/genetics-glossary/Protein
Holme,D.J and Peck,H. (1997) Analytical Biochemistry. 3rd
edition. (Addison Wesley Longman
Limited: London), pg 356-363
Nelson, D.L., Cox, M.M. (2005). Lehninger, Principles of Biochemistry. 4th
Edition (W.H
Freeman: New York), pg 116-120
Ruiz GA, Opazo-Navarrete M, Meurs M, Minor M, Sala G, van Boekel M, Stieger M, Janssen
AE. Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates
Obtained at Various Extraction pH. Food Biophys. 2016;11:184-197.
doi: 10.1007/s11483-016-9429-4. Epub 2016 Apr 23. PMID: 27212897; PMCID: PMC4851711.
Stryer, L., Tymoczko, J.L.,Berg,M.J. (1995) Biochemistry. 5th
Edition. (W.H Freeman and
company: New York) , pg 83
Voet.D (2015) Principles and Techniques of Practical Biochemistry. (Cambridge University
Press: UK), pg 132-150