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Int. J. Pharm. Sci. Rev. Res., 69(2), July - August 2021; Article No. 02, Pages: 19-24 ISSN 0976 – 044X
International Journal of Pharmaceutical Sciences Review and Research
International Journal of Pharmaceutical Sciences Review and Research
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19
Vaishali V. Acharya, Pratima Chaudhuri (Chattopadhyay)*
Molecular Biophysics Lab, Amity Institute of Biotechnology, Amity University, Sector-125, Noida, Uttar Pradesh-201313, India.
*Corresponding author’s E-mail: pchaudhuri@amity.edu
Received: 31-07-2020; Revised: 03-07-2021; Accepted: 12-07-2021; Published on: 15-08-2021.
ABSTRACT
Denaturation of protein is a biological phenomenon in which a protein loses its native shape due to the breaking or disruption of
weak chemical bonds and interactions which makes the protein biologically inactive. It is the process where properly folded proteins
formed under physiological conditions is transformed to an unfolded protein under non-physiological conditions. The process of
denaturation of proteins can occur under different physiological and chemical conditions. Denaturation can be reversible or
irreversible. Denaturation is mostly takes places when the protein is subjected under external elements like as inorganic solutes,
organic solvents, acids or bases, and by heat or irradiations. The denaturing agents or denaturants widely used in protein folding or
unfolding experiments are urea and guanidinium chloride (GdmCl). In denaturation, the alpha-helix structure and beta sheets
structure of the native protein are disrupted and unfolds it into any random shape. We can also say that denaturation occurs due to
the disruption of bonding interactions which are responsible for secondary structure and the tertiary structure of the proteins.
Keywords: Protein Denaturation, Denaturants, Urea, Guanidium Chloride, Protein Folding.
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DOI:
10.47583/ijpsrr.2021.v69i02.002
DOI link: http://dx.doi.org/10.47583/ijpsrr.2021.v69i02.002
INTRODUCTION
enaturation is a bio-chemical process which can be
defined as any alteration in secondary structure,
tertiary structure or the quaternary structure of
protein molecule which results in disruption of covalent
bonds. Almost all of the proteins known to exist, in their
native states, are folded into well-defined and are usually
essentially rigid and possess three-dimensional structures.
The changes occurring in the structure of protein is
generally partnered with alterations in properties like
chemical, physical and functional properties.1 It has been
found in many of the investigations that few proteins or
enzymes lose, their activities irreversibly or reversibly when
subjects to different natural or man-made conditions.
Glyceraldehyde Phosphate Dehydrogenase and Lactate
Dehydrogenase are example of two enzymes/proteins that
losses their properties when subjected to lower
temperature. Denaturation may be easily reversible, or it
may be “irreversible".
The changes associated with denaturation may include
ionization of carboxylic group, amino acid group, or the
phenolic groups. These may lead to rearrangements of the
molecules, followed by the release of sulfhydryl or
disulphide groups. These changes can lead to denaturation
or disruption of the overall protein molecule. And they may
also result in decreased protein solubility or loss of certain
biological activity of the protein.
Figure 1: Diagrammatic representation of denaturation of
protein.
Denatured states of the protein have become one of the
important areas of research in recent years due to their
importance in various phenomena like deciphering protein
folding problem and the molecular study of many diseases.
Denaturation of protein can be defined as intramolecular
change of the protein, which can be studied by studying
the displacement and relocation of the constituent groups
of the protein and atoms.
Various kinds of denaturation are generally followed the
below mentioned changes:
Reduced solubility: The process of protein denaturation is
often accompanied by denatured protein precipitation
through the addition of smaller amounts of neutral salts.
Decline trend in solubility of protein has been studied for
many years and is considered as one of the necessary
criteria for denaturation.
Modalities of Protein Denaturation and Nature of Denaturants
D
Review Article
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Decrease in biological activity: Some biochemical
activities of the proteins or enzymes may get destroyed
due to denaturation. The Proteolytic enzymes when
subjected to heat or treated with alkali become
inactivated. It is found that viruses lose their proteolytic
enzymes to produce disease, and it is also seen that certain
hormones lose their certain regulatory functions.
Depletion of crystallizing property: There are many
globular proteins which are capable for crystal formation.
Proteins lose its ability to crystalize on being subjected to
denaturation owing to alteration in structure and shape of
protein molecule.
Increased constituent group reactivity: Many alterations
in the chemical nature accompany the denaturation of the
protein.
“In few cases some of the constituents groups like
sulfhydryl, disulphide, or the phenolic groups are detected
after denaturation, but in the original protein state these
groups are either detected only to a small extent or not
detectable at all. The quantitative increase in these groups
generally relies on nature of the protein and on degree of
denaturation.”2
Alteration in shape of protein molecule: Protein
molecules in their native state are equipped with specific
molecular dimensions which are expressed as shape and
size. This molecular conformation may get affected due to
primary denaturation process. Using various methods, it
has been found that molar frictional ratio increases when
proteins are subjected to denaturation.
Susceptibility to enzymatic hydrolysis: A denatured or
unfolded protein molecule can be easily digested as
compared to native state of the protein by enzymes like
proteinases, which attack peptide bonds. So, from studies
it has been found that the process of denaturation makes
the protein molecule susceptible to proteolysis or the
protein molecule susceptibility increases towards
enzymatic hydrolysis.
Denaturation Degree: Characterization of protein
molecule is done by considering its amino acid
composition and physical configuration. Denaturation of
protein is mainly due to the change in its physical
conformation of polypeptide chains inside protein
molecule. Denaturation Degree can be measured by
studying the degree to which the structure of protein has
been altered. The denaturation degree in protein structure
relays on the nature of protein as well as on the nature of
the denaturing agents.
MECHANISM OF PROTEIN DENATURATION
Denaturation is considered to be a tool used for probing the
folding properties of the proteins. In recent time,
denatured state of proteins has received much importance
due of its active participation in study for understanding the
protein folding process.3 Denaturing agents mostly used in
the folding and unfolding experiments of protein are urea
and guanidinium chloride (GdmCl).
The challenges faced in this experiment was whether one
can define the denaturation process without
denaturants/denaturing agent or not. The experiment
done by Privalov and colleagues provided an answer to
this. The experiment concluded that thermodynamic
properties of unfolding of protein do not depend on the
denaturants but structural properties of the protein
depends on the denaturants. It can be also mentioned like
the net or total enthalpies and entropies of the protein
denaturation are intrinsic properties of proteins.
Figure 2: Diagrammatic representation of protein
denaturation using GdmCl and Urea as denaturants. 4
The mechanism of folding and unfolding of protein is
generally studied through thorough analysis of structure of
the protein and not through direct calorimetric
measurements. Like the study of secondary structure is
done by circular dichroism, burial of tryptophan is studied
by fluorescence, and so on.
The molecular study for protein denaturation using urea
and GdmCl as denaturants does not have many strong
evidences yet. For this two models was proposed to study.
One of the two model is based on a direct, interaction
between the denaturant and the protein and the other
model is based on the modification of hydrogen-bond
structure of water resulting in reduction of strength of
hydrophobic interactions.
Protein Denaturation Using Urea: Protein denaturation
using Urea as denaturant was first studied in the early
years the past century. During 1930s, Urea was most
widely and commonly used osmolyte for the study in
folding-unfolding of protein i.e., denaturation of the
protein.5 In research studies, it was found that urea helps
in denaturation either by directly interacting with protein
initiating solvation of polypeptide chain by water and urea
or indirectly by modifying the water molecule structure
resulting in changes in the behavior of solvent which
weakens or reduces the hydrophobic effect.
Most versions of interaction model states that urea favors
the unfolding by binds to the protein, and stabilizes, the
denatured state. But the drawback of this interpretation is
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21
that it does not provide explanation for how the protein
itself overcomes the kinetic barrier resulting unfolding of
the protein.6
From a study conducted on the “Kinetics of RNase A at Low
Urea Concentrations”.5 It was concluded that urea
inhibition of RNaseA adheres to uncomplicated
competitive model that suggests the probability that
additional product is formed by this compound with
residues present at active center of the enzyme.5 Wu and
Wang suggested that the concentrations of urea utilized
were very less than pre-transition region concentration for
the unfolding. This study also has put light on the fact that
in aqueous urea solutions RNase can be inhibited
competitively.
Protein Denaturation by GdmCl: Guanidinium cation was
found to be the most effective chemical reagent or
denaturant which reduces stability of protein when it is
added to the aqueous solvent. Two mechanisms are found
by which denaturant can destabilize proteins. One of these
mechanisms is indirect method by modifying solvent
properties of the water and the other is direct method
which takes place by specifically binding with the groups of
protein. The indirect denaturation mechanism generally is
not applicable for Gdm+.7 Various experimental evidence
proves that the interaction is very weak between Gdm+
and water molecules. Gdm+ was found to affect the
interactions between water molecules in the hydration
shell of other solutes. Gdm+ enhances solubility of
nonpolar groups in proteins molecule as it lacks hydration
shell. Gdm+ denatures proteins by interacting with the
protein molecules directly it does not bind peptide groups
using hydrogen bond.8
METHODS OF DENATURATION USING DIFFERENT TYPES
OF DENATURANTS
Physical Agents:
Heat/High Temperature: If any solution of protein is
subjected to heat neat to its isoelectric point, the protein
will coagulate. If the temperature is raised to ten degrees
this heat coagulation of protein takes place about 600
times faster. Heat is consider as most familiar denaturing
agent for protein. Heat denatured protein has an increased
susceptibility for aggregation, depending on pH, dielectric
constant and ionic strength of the medium. Acid and alkali
dissolve heat coagulated protein and heat coagulated
protein can be dissolved even at the isoelectric point by a
number of substances such as urea, guanidine
hydrochloride, detergents, and salicylate. The protein
denatured by heat is slowly regains its original soluble form
when cooled. Non-reversible denaturation is caused by
heating for longer duration, heating of the protein solution
for shorter duration at its isoelectric point or by adding salt.
There are many enzymes which shows thermophilic
properties i.e. they show stability in higher temperature.
“The most common stabilizing agents at high
temperatures are probably di-myo-inositol 1,1«-phosphate
and cyclic 2,3-diphosphoglycerate. These enzymes are
present in Pyrococcus woesei and Methanothermus
fervidus respectively. These agents increase half-lives of
some the enzymes by around 130-fold when subjected to
90 °C in presence of potassium”.9 Daniel et al. have studied
that the tertiary structure of proteins are not fully stable.9
Stability of enzyme at high-temperature was performed by
them by subjecting the enzyme to heat then rapidly
cooling it and assaying it at lower temperature for residual
activity. The stability of the enzyme at higher temperature
is studied by heating the enzyme at higher temperature
and then rapidly cooling it and then subjecting it to the
assaying for residual activity at low temperature.
Pressure: Under high pressure the hydrophobic interaction
of the protein weakens and it kicks starts the process of the
denaturation of the protein. Moderate pressure cannot
drive the unfolding of the protein though some structural
changes can occur due to which protein can lose its
activity.10
The effect of pressure on protein denaturation has been
studied less. Neurath et al. has studied that denaturation
of proteins can occur at high pressures of approximately
6000 kg / cm2, and the protein coagulation can take place
at the pressures of approximately 10,000 kg / cm2.2
Freezing/Low temperature: For many years’ studies have
shown that proteins losses that activity when subject to
low temperature or stored in refrigerator temperature.
The rate and extent of denaturation appear to be affected
by salt concentration and pH and also by freezing
temperature. This type of cold inactivation or cryo-
inactivation has two grounds; first of them is cold
dissociation and the other type is cold inactivation of
proteins and enzymes. Cold dissociation is generally for the
oligomeric proteins. These enzymes when subjected to low
temperature and dissociate in the lower association level
and the activities of original oligomeric structures are lost.
The changes are mostly reversible and therefore
incubating at the room temperature for longer duration
will make the protein regain activity by restoring native
oligomeric structures. Cold inactivation of the enzymes and
the proteins, is the process observed in dimeric or
monomeric globular proteins. It is also observed in situ,
where dissociation process is not involved as the primary
step.11 This kind of protein inactivation is also called or
termed as cold denaturation. The denaturation of various
globular proteins subjected under low temperature have
been studied. In a research, it was stated that yeast prion
protein Ure2 showed cold denaturation below 35 degree
Celsius at 200 Megapascal and was more susceptible to
cold denaturation at lower ionic strength. The cold
denatured state of the protein is studied by various types
of spectroscopic methods, like UV absorbance,
fluorescence, IR and NMR, and the scattering methods
including SAXS and light scattering.
Irradiation: If the protein molecules are exposed to
ultraviolet light it causes coagulation of the protein.
Coagulation of the proteins using ultraviolet radiation
consists of mainly two methods: 1) proper light
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22
denaturation, 2) photochemical reaction which does on
depend on temperature and then it is followed by the
flocculation of denatured protein. This coagulation of
protein has high temperature coefficient. Irradiation can
cause change in the state of protein solutions aggregation
which can lead to loss of biological activity and also the pH
of solutions tends to shift towards the isoelectric point of
protein.
Sound waves: When protein molecules are subjected to
the mechanical vibrations of high intensity, produced by
ultrasonic or sonic waves, the enzymes losses their activity
and proteins are coagulated.
Surface Forces: If the proteins are spread over an aqueous
surface, or in an interface, protein denaturation takes
place by protein molecules unfolding into structures which
resembles fully uncoiled polypeptide chains.
Chemical Agent:
Acid/Low pH: It has been found that some proteins retain
their native conformations at very low pH if the
temperature is kept low whereas the others undergoes
unfolding. The decrease in pH/low pH can cause
conformational change in the second type of proteins.
Many proteins lie intermediate between these extremes.
Lysozymes is one of the protein which remain unaffected
in low pH at room temperature. β-lactoglobulin and
ribonuclease are also the examples of protein resistant to
acid denaturation. Yeast glyceraldehyde-3- phosphate
dehydrogenase is a protein which undergoes significant
change in enzymatic activity, molecular weight at low pH
and low temperature. It shows transition between pH4-pH
11 but highly cooperative transition occurs below pH 4, it
results in loss of enzymatic activity, dissociation into
subunits, and thorough disorganization of the
conformation of the subunits themselves. Ferrimyoglobin
is highly unstable at low pH. When it is subjected to pH 5 and
4 at room temperature undergoes a transition. The
denatured state is probably the same as that of the product
of the reversible thermal transition of this protein, a
conclusion based primarily on the thermodynamic analysis
of Acampora and Herman (1967).12
Alkali/High pH: Some proteins shows denaturation at
alkaline pH is like that at acid pH while in other proteins
the course of alkaline denaturation is different.
Hemoglobin and Myoglobin stable toward alkaline pH than
toward acid pH. The denaturation of proteins at alkaline pH
is complicated for proteins that contain thiol groups or
disulphide bonds. Apart from chemical modification, the
products of alkaline denaturation are undoubtedly as
diverse as the products of acid denaturation. Because
buried tyrosyl residues tend to become exposed above pH
10 or 11, there may be less tendency to retain residual
structure at very high pH than at very low pH, but
meaningful experimental studies are difficult to make
because of the prevalence of chemical instability.
Organic Solvents: Denaturation or coagulation of protein
could be seen when alcohol or acetone are added to
protein solution aqueous in nature in region of isoelectric
point. This procedure is dependent on the temperature
and not appears to happen at a quantifiable rate at
temperatures underneath - 15°C.
Studies had reported that proteins which belongs to group
prolamins like hordein, secalin and kafirin need alcoholic
medium for disintegration of protein while no indication of
denaturation getting obvious.
It is likely that organic solvent denaturation is associated
with the impact of conclusion on dielectric constant of
medium; however there is an incomplete definitive proof
concerning the underlying mechanism.
Alcohols and glycol can be good denaturant. The branching
of hydrocarbon in alcohol tends to decrease their
denaturing properties. As denaturants, glycols are less
efficient than the corresponding alcohols. It suggests that
increased polarity or hydrogen-bonding capacity is of
secondary importance when compared with the effects of
increasing hydrocarbon content.13
Organic solutes: There are several outstanding organic
compounds acts as denaturing agents. Chemical and
physical method has been deliberately studied to observe
the activity of acetamide, urea, formamide, and guanidine
salts present in concentrated solutions.
The above-mentioned organic denaturants have increased
the dissolvability of the denatured protein, subsequently
offering especially appropriate conditions for examining
the procedure healthy by aggregation or flocculation.
Impact of organic denaturing compounds on the solubility
of denatured protein could be observed, subsequently
provides appropriate conditions to study procedure
unimpaired by flocculation or aggregation of denatured
protein.
The recent investigation on protein denaturation has been
conducted on studying the denaturing effects of the
synthetic detergents. As per studies conducted on the
anionic detergents like alkyl sulfonates and alkyl sulfates,
alkyl sulfosuccinates and mixed alkyl-aryl sulfonates, and
cationic detergents like alkyl-aryl-substituted ammonium
halides were efficient denaturants.2 The powerful
denaturing action of these denaturants surpasses even the
denaturing activity of GdmCl of equimolar concentrations.
THERMODYNAMICS OF DENATURED STATE LOOP
FORMATION
A simple loop is the most common structure obtained from
the disordered or denatured protein. The conformational
constraints that helps in loop formation and also control
the folding efficiency of protein; because of this they can
provide information on basis of misfolding diseases. Till
date various methods have been devised to study loop
formation kinetics. The evidences from the previous
studies were used to explain a regulatory check for protein
folding. In a research study it was found that loops are
formed under denaturing conditions when at sixth
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23
coordination of heme site is taken by unique histidine.14
The below diagram is taken from the research study on
“Thermodynamics of denatured state loop formation” by
Bowler.15
Figure 3: Diagrammatic representation of loop formation
of histidine and heme.15
Simple pH titration is used to measure stability of loop.
Altering the sequence position in engineered histidine can
control the size of the loop subjected under denaturing
conditions
Dependence of stability of loop on loop size can be used to
evaluate factors like stiffness of chain and degree of
representation of chain as a random coil. In many research
studies for DNA loop formation large scaling exponents
have also been observed. The thermodynamic methods
employed for studying the formation of denatured state
loop have provided important information about the
conformational constraints. It is evident from kinetic
methods that smaller loops form faster, equilibrium
studies have proved that the smallest loops are always not
most stable and thus not necessarily that it will lead to a
productive folding.
CONCLUSION
The mechanism of protein denaturation has been studied
in this review paper. It would enhance our knowledge on
the events of protein folding and unfolding by giving
detailed analysis of the steps of this fundamental process
associated with the structural stability of the proteins.5 For
analyzing or deep understanding the denaturation of
proteins various types of spectroscopic methods, like UV
absorbance, IR, fluorescence, Nuclear Magnetic Resonance
and the various scattering methods includes SAXS and light
scattering can be used. These methods are known to
scrutinize the behaviors of different segments and the
different levels of protein denaturation.
Uncovering residual/unused structure of the protein in its
denatured states have been playing a vital role in research
studies for characterization of thermodynamic significance
of the residual structure. Thermodynamic methods are
vital for studying how the protein folding process in
impacted by factors like compactness of protein structure,
excluded volume and internal friction present in the
protein chain.
It has been mentioned in research papers of different
researchers that relation between the kinetics of protein
folding and stability of the denatured protein state
structure is an integral part to examine how the protein
folding efficiency is impacted by the denatured state of the
protein.
Thus, denatured states of protein possess various
interesting as well as unusual characteristics and
properties which are very important to understanding
folding of protein molecules and its stability.
Several future insights and directions are very much
evidential from above mentioned text. Studying the role of
non-native against native protein is important for
conducting various studies. The quantitative
measurements for strength of electrostatic interactions in
denatured state have been conducted. Likewise,
measurements of hydrophobic interactions are also
required, particularly for those which are modulated by
aromatic molecules like tryptophan.
In the field of protein engineering, the techniques for
combining native and denatured state would be useful in
protein stabilization for development of hyper stable
proteins. The recent advancement in research studies of
the thermodynamics of denatured state provides an
excellent foundation many future researches.
Denatured state of protein or the denatured protein is
biological state which consists of the distribution of various
molecular conformations and the average of these are
analyzed or quantified by performing various experiments.
There are many evidences which states that even in highly
effective denaturants like 6M GdmCI and 9M urea, some
structure may continue to exist in its native protein chains.
It is studied under the physiological conditions, denatured
states of almost all proteins appear to be very compact
with large number of secondary structure. Various
theoretical as well as experimental studies has suggested
that entropies of chain conformational, hydrophobic
interactions and electrostatic forces plays a vital role in
determining the structure. The process of protein
denaturation in urea or GdmCI could be modelled as a two-
state transition between the original or native structure
and a relatively compact denatured state. It experiences a
gradual increase in radius if denaturant is added to it
further. When a protein gains large net charge in acids or
bases, it tends to exists in the two stable denatured form,
one compact structure and other one is extensively
unfolded.
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