Full lab Report on

Exercise No. 4
Protein Denaturation

Falo, Ma. Elaine F.

CHEM 161.1 1L
2nd Semester 2015-2016

Groupmates:
Abulencia, Anabel
Dequina, Peter
Lebbe, Rehana

Date performed: February 11, 2016

Date Submitted: February 18, 2016

Mr. Bong Carlo N. Remillon

I.

Introduction

Proteins are considered as the most abundant among the bio-organic molecules. These are
polymeric compounds composed of its monomers which are the twenty standard amino acids. These
monomers are connected by an amide bond called peptide bond in order to form a linear chain of amino
acid. Proteins can be classified based on their composition such as simple proteins and conjugated
proteins. Simple proteins will yield only amino acids or their derivatives when hydrolyzed while
conjugated proteins are simple proteins combined with non-protein parts such as nucleic acids,
carbohydrates, lipids, phosphate groups and others. Also, they perform varied functions in the biological
system such as for transport proteins, enzyme (catalysts), storage of nutrients, structural or mechanical
support, motile proteins, defense proteins and regulatory proteins. Proteins can only perform these
functions when they are at their native structure. There are four structure that define the three dimensional
shape of the water soluble proteins which makes it in its native structure. Although there is a complexity
in proteins, there must be hierarchy or levels of organization.
The basic structure of a protein is the primary structure. In the primary structure, it gives the basic
knowledge of the amino acid sequence of a polypeptide chain. It bears the shape and final structure
therefore it has to be stabilized. The stabilizing factor of the primary structure is the peptide bond. A
peptide bond is formed by linking the

-carboxyl group of one amino acid with the

-amino

group of another amino acid. This peptide bond is quite kinetically stable due to extremely slow rate of
hydrolysis in which it can last for 1000 years without the presence of a catalyst (Stryer, Berg &
Tymoczko, 2013). Also, another reason why peptide bond serves as the stabilizing factor of the primary
structure is because of its rigidity because it is resonance stable and its 40% double bond character. Its
polypeptide chain has a sense of directionality wherein the amino terminal is the beginning or the basic
convention (N-terminal

C-terminal). The polypeptide has a backbone which is the

-carbon,

acyl carbon and the amide nitrogen. Although they have the same backbone structure, there are different
proteins since they differ in the identity of R groups in the amino acid residues. The proteins primary
structure is important because it determines the tertiary structure and the tertiary structure, on the other
hand, determines the proteins function.
Secondary structure is the regular and repetitive in which the polypeptide folds as a result of
hydrogen bonding and other weak forces without regards to the conformation of its side chains. There are
two types of secondary structure such as the

-helix and

-pleated sheets. In the

-helix, the

main stabilizing force is the intrachain hydrogen bonding between the NH and CO groups of the main
chain about four amino acid residues away. All the peptide bonds are used in H-bonding to form an
extensive H-bond formation. Almost all helices are right handed. On the other hand,
are stabilized by hydrogen bonding between polypeptide chains. Adjacent

-pleated sheets

sheets can run in the

opposite directions (anti-parallel) or in the same direction (parallel). About the C-N bond, the
conformation that is more preferred is trans. The ramachandran plot gives an idea on what is the possible
2o structure of the polypeptide. The structure of each amino acid in a polypeptide can be adjusted by

rotation about two single bonds (Voet, Pratt & Voet, 2012). The torsion angles
between the

-C and carbonyl carbon while is the rotation between the

which is the rotation

-Carbon and amide

nitrogen. Not all the combinations of psi and phi are possible due to steric clashes. The only possible
combinations are given by the Ramachandran diagram.
Tertiary structure is the three dimensional structure resulting from the interaction of the side
chains or R groups. There are four stabilizing forces which are the hydrophobic interactions, ionic
interactions, disulfide bonds and the hydrogen bonding. This structure is the highest level common to all
proteins since not all proteins have more than one polypeptide. The tertiary structure is also created when
the molecules fold back themselves outside of the helical segments which direct the hydrophobic (water
repellant) portions to the interior and the hydrophilic (water loving) portions on the exterior. This makes
the protein water soluble. The highest level of the protein organization is the quaternary structure. This is
only when there is more than one polypeptide chain present. Its stabilizing factor is the same as that of
tertiary structure.
Proteins are said to be in its native conformation when it is stable and exhibits biological activity
in terms of its specific combinations of the secondary, tertiary and quaternary. Any deviation from this
will affect the proteins ability to perform its specific function or biological activity. Protein, is then said to
be denatured. Denaturation is said to be the any change that results in the disruption of the secondary,
tertiary and quaternary levels of the protein. The primary structure still remains intact because of the
resonance stabilized peptide bond. The effect may be permanent or temporary depending on the extent of
the denaturing agent.
The concentration of protein in a protein isolate can be determined using spectrophotometer
especially when the protein contains chromophore or by the existence of the aromatic rings in the side
chains of amino acids which makes the polypeptides absorb strongly at the UV region.
In this experiment, the effect of the physical and chemical agents is observed on the protein
isolate from spirulina tablet which is phycocyanin. Also, its purity and concentration will also be
determined using the absorbance obtained at 620nm, 650nm and 280nm.
II.

Methodology

Preparation of sample
Two spirulina tablets are weighed and approximately the same amount is weighed for fine silica.
Using mortar and pestle, these two are grinded against the sides and bottom of mortar to ensure that the
maximum breakage of the cell walls until a fine dark green powder mixture is obtained. For the use of the
class, 30mL 0.1M phosphate buffer pH 4.7 is added to the mixture and is mixed. After mixing, the
mixture is transferred to the eppendorf and mixed using vortex mixer. Another tube with distilled water,
for centrifugation, is prepared with the same amount as that of the protein mixture. It is placed in the
centrifuged for 2 minutes with 1000rpm. The supernatant is transferred to 125mL Erlenmeyer flask.
Denaturation of proteins

Eight test tubes are prepared and are labeled as 1,2,3,4,5,6,7 and 8. Each of them is placed with
0.5mL of the protein solution. For test tube 1, it is added with 1.0mL of 6.0M HCl. For test tube 2, it is
added with 1.0mL of 2.0M NaOH. For test tube 3, it is added with 1.0mL of 0.2M Lead Acetate. For test
tube 4, it is added with 1.0mL of 10% trichloroacetic acid. For test tube 5, it is added with 1.0mL of
acetone. On the other hand, test tube 6 is placed in a hot water bath while test tube 7 is placed on a cold
water bath. Test tube 8 served as the control.
Determination of purity and concentration of the protein isolate
In order to determine the concentration of the isolate, the absorbance is measured using
spectrophotometer at wavelengths 620nm and 650nm. The absorbance is also measured at 280nm for the
determination of the purity of the isolate if it is of analytical or food grade.
III.

Results and Discussion

In this experiment, the protein which is used is obtained from spirulina tablets. Spirulina is a
commercial name which refers to dried biomass of cyanobacterium, Arthrospira platensis. It is blue-green
algae which is edible for humans because its cell wall does not contain cellulose. Humans do not have the
enzyme which can digest or break down cellulose. This is the reason why spirulina is used as a food
supplement. Also, it has large percentage of protein contents compared to carbohydrates or fats.
Phycocyanin is one of the major pigment constituent of Spirulina. It is water soluble blue pigment.
Spirulina consists of two biliproteins which includes c-phycocyanin and allophycocyanin. The
chromophore is phycobilin which is an open tetrapyrrole (Vonshak,2002).

Figure 4.1. Chemical structure of phycocyanin bilin chromophore.

Source: Datla, P. The Wonder Molecule called phycocyanin.

Table 4.1. Observations on the denaturation of phycocyanin upon exposure to denaturing agents.
Test Tube No.
1

Reagent added/Conditions
6.0M HCl

2.0M NaOH

0.2M Lead acetate

10% Trichloroacetic acid

Acetone

Observations
The solution turned from blue to
light green.
The solution turned from blue to
light yellow.
There is a formation of white
precipitate.
The solution turned from blue to
turbid light blue.
There is a change from blue
solution into white turbid mixture.

Hot water bath

7
8

Cold water bath

control

The solution turned to light yellow

solution.
It is still blue solution.
It is still blue solution.

It can be observed that upon addition of different reagents or employing some changes
in conditions, there is an effect in the native state of the protein. The changes can be observed by the
change in color and the formation of precipitates. In the first and second test tubes, the denaturing agents
added are strong acid (HCl) and base (NaOH). There is a change in color in the first and second test tubes
indicate that the phycocyanin has been denatured. Its ultraviolet (UV) light absorption has been altered.
Strong acids and bases usually interrupt the hydrogen bonding and the ionic interactions in the protein.
Upon adding an acid it can protonate some of the R groups of an amino acid and adding base will
deprotonate some of the amino acid which in turn can cause the side chains to not be able to participate in
hydrogen bonding. The hydrogen bonding and salt interactions which holds the tertiary structure will be
broken and the protein will coil randomly losing its functions. It is not only in the change in color that a
protein can be considered denatured but also in the formation of precipitate which can be seen upon the
addition of lead acetate. Lead (Pb2+) is a heavy metal ion which has high attraction for carboxylate groups
and sulfhydryl groups in proteins. It affects the ionic interactions and it forms metal complexes with the
protein which precipitates out.
The next denaturing agent employed is the trichloroacetic acid. This reagent is
commonly used for protein precipitation since it readily denatures proteins upon addition or shifts the
condition so that the protein is not active, though not necessarily denatured (Scopes, 1994). However,
based on the observations on the phycocyanin isolate, there is a change in color from blue solution to light
blue. It is close to the original color of the protein although its UV absorption has also been altered,
somehow. The negatively charged tricholoroacetate ions induce denaturation by disrupting the
electrostatic interactions that stabilize the native conformation of proteins. The non-polar part of the
protein then becomes exposed. For the fifth test tube, acetone is added and produced a turbid mixture.
Acetone is an organic solvent which disrupts hydrophobic interactions. It can insert itself between the side
chains of hydrophobic amino acids. Also, it interrupts the hydrogen bonds due to the C=O of the acetone.
Among the changes in the conditions employed is the difference in temperature. Test
tube 6 is place at hot water bath while test tube 7 is placed at cold water bath. When placed at a high
temperature, the protein is provided by energy which is relatively enough to break the hydrogen bonds,
ionic interactions and the hydrophobic interactions the holds the protein in its folded native state. The
protein then starts to uncoil randomly and lose its biological functions. Just like the other denaturing
agents, it has disrupted the forces that stabilize the quaternary, tertiary and secondary levels but not the
primary structure. It is because of the stable peptide bond which remains the amino acid sequence of the
protein still intact. Although most of the denaturation is irreversible, some can be renatured under
laboratory conditions such as ribonuclease which upon denaturation of 8M urea, renaturation occurs upon
dialysis and the enzyme activity is restored.
It can be observed that when test tube 7 is compared to that of the control test tube (8)
there is no difference in the color. Since there are no observed changes in test tube 7, it can be said the
protein isolate is in its native form and did not undergo denaturation upon placing it in cold water bath

which means that the phycocyanin is stable at low temperatures. In a journal article entitled Stable
Isolation of Phycocyanin from Spirulina platensis Associated with High-Pressure Extraction Process, the
conditions high pressure and low temperature favored the extraction with increased percent yields since it
achieved the destruction of the Spirulina platensis cell membrane without denaturing the phycocyanin
which is composed of temperature sensitive

and

sub-units. It can be concluded that the

phycocyanin isolate in this experiment follows the same behavior as that sample in the journal article.
Table 4.2. Data on the absorbance of the phycocyanin isolate on different wavelengths for the calculation
of the purity and concentration.
Wavelength (nm)
620
650
280

Absorbance
1.202
0.490
1.78

Studying proteins in vitro requires the isolation of the protein from the natural source and keeping it in
conditions which maintains its function. It is important that the isolated protein is purified to determine its
biological activity or to determine its amino acid sequence. From the given data on the absorbance, the
concentration and purity of the protein isolate can be calculated. For the concentration, it can be
calculated using the formula
CPC (mg/mL) =

[ A 6200.70 ( A 650 ) ]
7.38

The concentration of the isolate is 0.1164 mg/mL while the purity is 0.6753 which means that the sample
cannot be considered food grade since it is not exactly 0.7 in value. The purity is determined by the ratio
of the absorbance at 620nm and absorbance at 280nm. A sample with a ratio of greater than 4.0 means
that it is analytical grade while a ratio of 0.7 means that the sample is food grade. Theoretically, the
phycocyanin is said to be food grade since it is edible for human consumption as a supplement.
Protein denaturation also has clinical application. One example is the characterization of
denatured proteins due to its arising importance. In one of the research activities of Center for protein
chemistry by University of Texas, the conformational changes brought about by denaturation is said to be
the cause of many neurodegenerative diseases such as Prions disease, Parkinsons disease and
Alzheimers disease. Being able to elucidate or study the conformational isomers of the denatured protein
can have a potential use in disease diagnosis and treatment. The Center for protein chemistry has
developed ways on how to isolate the denatured proteins for further studies. Initial studies show that the
X isomers (scrambled structures of disulfide containing proteins) enhance aggregation and increase
immunogenicity compared to the native protein (Jiang, nd). Also, in blood analysis, the denaturation
property of protein is used to eliminate the proteins of blood.

IV.

Sample Calculations

For the calculation of phycocyanin concentration:

CPC (mg/mL) =

[ A 6200.70 ( A 650 ) ]
7.38

[1.2020.70 ( 0.490 ) ]
7.38
= 0.1164 mg/mL

For the calculation of the phycocyanin purity:

A 620
A 280 =

1.202
1.78

= 0.6753
V.

Summary and Conclusions

Upon conducting the experiment, the different reagents added to the phycocyanin isolate
exhibit different changes caused upon by denaturation. Each denaturing reagents disrupts specific
stabilizing forces that keep the protein in its natural or native folded form. The denaturing agents can be
classified as physical or chemical means. The strong acid (HCl) and base (NaOH) produced a change in
color in which these reagents affects the hydrogen bonding and the ionic interactions. The heavy metal
ion, Pb2+ in lead acetate, caused precipitation of white solids. This reagent affected the electrostatic or
ionic interactions which resulted in precipitation of the metal complexes. Acetone, an organic solvent,
disrupted the hydrophobic interactions in the protein as well as the hydrogen bonding due to the presence
of C=O. On the other hand, trichloroacetic acid (TCA) is usually used for protein precipitation and for
partial denaturation. There is a change in color upon addition of TCA which can be concluded that the
protein deviated from its natural property and it means that it might have as well lost its original
biological function. It cannot be concluded if the phycocyanin only underwent partial determination.
From the employed difference in temperature, the phycocyanin underwent denaturation when placed in
hot temperature since it provided the protein energy to destroy the hydrogen bonds and ionic interactions
which holds the proteins structure. It can be concluded that the phycocyanin isolate is stable at low
temperatures since there is no observed changes when compared to the control test tube.
The phycocyanin isolate has a concentration of 0.1164 mg/mL and a purity of 0.6753
which is food grade.
VI.

References/Literature Cited

Datla, P. (2011). The wonder molecule called phycocyanin. India: Parry Nutraceuticals,
Division of EID Parry Ltd.

Jiang, C. Production of stabilized non-native protein isomers and characterization of

their biological and immunological properties. Retrieved from https://www.uth.edu/imm/centers/proteinchemistry.htm
Scopes, R. (1994). Protein Purification: Principles and Practice. Third Edition. New
York: Springer Science & Business Media
Seo, Y.C., Choi, W.S., Park, J.H., Park, J.O., Jung, K.H. & Lee, H.Y. (2013). Stable
isolation of phycocyanin from Spirulina platensis associated with high-pressure extraction process.
International Journal of molecular sciences, 14(1), 1778-1787.
Stryer, L., Berg, J.M. & Tymoczko, J.L. (2013). Biochemistry A short course . Second
Edition. USA: W.H. Freeman and Company
Voet, D., Voet, J.G. & Pratt,C.W. (2013). Fundamentals of Biochemistry. Fourth Edition.
USA: John Wiley & Sons, Inc.
Vonshak, A. (2002). Spirulina Platensis Arthrospira: Physiology, Cell-Biology and
Biotechnology. USA: CRC Press