Protein.pdf

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PROTEINS i lai
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PROTEINS AS THE MAJOR COMPONENTS OF ‘THE BODY
» Proteins are high-molecular nitrogen-containing organic compounds which
_, "are polymers of amino acids. The natural proteins are composed of 20 amino acids,
bound to each other with peptide bonds. These 20 amino acids may be joined In
dificrent sequence and in many different combinations. The variety in proportion
oi amino acids and diversity in their combination (sequence) produces wast
diversity of proteins in the body.
In the nature, 10" to 10 ' diverse proteins are present and they provide for
«vistence of about 10° species of living organisms, from virus to human. Human
organism numbers over 50,000 proteins, and each protein has distinctive structure
and function.
The major constituents of proteins are carbon, hydrogen, oxygen, and
nitrogen atoms; the minor constituents are sulfur and phosphorus. The average
content of different atoms in proteins is shown in Table 1.1.
Table 1.1. Elementary content of proteins
Atoms I Average content (%) |
C 52 (50-55)
| o) | 22(21.5-23.5)
: H 7(6.5-7.3)
N | 16 (15-17)
s | 0.5
Proieins are the most abundant biological molecules occurring in all cells
and have paramount importance.
for the organism. Proteins account for 25 % of wet
body weight and 45 % of dry weight. The maximal concentration of protein is in
skeletal muscle, liver, spleen, and kidney.
Proteins are the molecular instrument through which genetic information
is expressed.
Proteins are used for body building. All the major functions of the body
are carried out by proteins. Thus, proteins constitute a basis for both structure
and functions of the living organism. +oasy
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FUNCTIONS OF PROTEINS ene vid miles ~
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Proteins are the principle functional elements of the body. They are “alive”
or “animated” molecules wh.ch exhibit numerous biological functions.
| Structural funcfion, Proteins serve as building blocks for cellular and
extracellular structures. The proteins that perform this function are the most
widespread quantitatively in 112 body. E.g. proteoglycans (complexes of proteins
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oF F Avith carbohydrates) and collagen in connective tissue; a-keratin in hair skin ane
RY nails; elastin in vascular wall, and membrane proteins.
) x +" 2. Contractile function. The proteins involved in muscle coniracuon ars
"represented by actin, myosin, tropomyosin, and troponin.
og 3. Catalytic function. This function is performed by enzymes that are
NG biological catalysts.
¢ Transport function. Transport of oxygen and carbon dioxide is carried
out by hemoglobin (in the blood) and myoglobin (in the cell). Albumins
: C participate in transport of fatty acids, bilirubin and other water-insolubic molecules
= Jin the blood. Transferrin transports iron, ceruloplasmin transports copper in the
"=" ‘blood plasma.
A 5. Nutrition function. Reserve proteins such as ovalbumin in the egg white.
’ or casein in milk perform this function. Proteins of blood plasma and other tissues
| May serve as an amino acid reserve. Due to degradation of proteins in the liver.
blood plasma, muscle and intestinal mucosa under prolonged starvation. amino
acids are released which ensures synthesis of essential proteins. enzymes,
hormones, etc.
6. Clotting function. Certain blood plasma proteins are major components of
the blood coagulation system.
7. Protective (defence) function. Immune system produces the specific
protective proteins (antibodies or immunoglobulins) in response to the i
invasion
of the body by bacteria, toxins or viruses. Proteins of the blood coaguiation system
(fibrinogen etc.) result in the formation of a clot which stops bleeding and
prevents the organism from the blood loss.
8. Regulatory function. A number of hormones are proteins, and regulate
biochemical reactions and function in the body. Receptors (proteins conjugated |
with carbohydrates) participate in recognition of certain specific hormones.
9. Maintenance of oncotic pressure. Proteins maintain the oncotic pressure
in the blood and provide thereby for a constant blood volume in the organism.
10. Maintenance of constant blood pH level. Proteins form a buffer svsiem
which maintains the acid- base balance in the blood plasma.
STRUCTURE OF AMINO ACIDS
The general formula of amino acids is shown in Fig. 1.
Fig. 1. General formula of amino acids.

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~~ Each amino acid has a central carbon, called a-carbon, to which four
different groups are attached: hydrogen atom (-H), a basic amino group (-NH,), an
acidic carboxyl group (-COOH), and a distinctive side chain radical (-R). he
All the proteinogenic amino acids have an amino group attached to the
carbon atom next to carboxyl group, i.e. in the o-position; that is why they are
called as o-amino acids.
The specific structural and functional diversity of protein molecules is based
on the chemical structure and physicochemical properties of amino acid radicals.
These radicals provide chemical individuality of proteins and their unique
functions never encountered in other biopolymers.
STRUCTURE OF AMINO ACIDS
CLASSIFICATION OF AMINO ACIDS
Amino acids are classified in various ways.
1. Classification based on nutritional requirement:
a) essential amino acids: threonine, methionine, leucine, isoleucine, valine,
* lysine, arginine, phenylalanine, tryptophan, and histidine;
b) non-essential amino acids: glycine, alanine, serine, cysteine, aspartate,
asparagine, glutamate, glutamine, tyrosine, and proline.
2. Classification based on the number of amino and carboxyl groups
present in the molecule:
a) monoamino dicarboxylic amino acids: aspartate (aspartic acid);
glutamate (glutamic acid);
b) diamino monocarboxylic amino acids: lysine, arginine;
c) monoamino monocarboxylic amino acids: all the others.
3. Classification based on the structure of the radical:
aliphatic amino acids: glycine, alanine, valine, leucine, and isoleucine; all
these amino acids are hydrophobic in nature;
byparomatic amino acids: phenylalanine, tyrosine, and tryptophan;
c) sulfu r-containing amino acids: cysteine and methionine;
d) hydroxy amino acids: serine, threonine, and tyrosine (the latter is also
aromatic amino acid);
e) imino acid: proline;
f) amides of amino acids: asparagine and glutamine.
4. Classification based on the polarity of the radical:
a) non-polar or hydrophobic amino acids: glycine, alanine, valine, leucine,
isoleucine, methionine, proline, phenylalanine, and tryptophan; the part of a
protein made up of these amino acids will be hydrophobic in nature; .
b) polar (hydrophilic) amino acids with non-ionic (non-charged) side
chains: serine, threonine, cysteine (has low polarity), tyrosine, glutamine.
asparagine;

ATL Steps of hydrolysis
Proteins
High molecular polypeptides
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Low molecular polypeptides »
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Oligo-, tri-, and dipeptides
Amino acids
The side chain of tryptophan is almost completely degraded by acid
hydrolysis, and small amounts of serine, threonine, and tyrosine are also lost. In
alkali hydrolysis, almost all the amino acids are destroyed, but not tryptophan. The
end products of both acid and alkali hydrolysis, joined together, form a complete
“mixture of 20 amino acids (protein hydrolysate).
Hydrolysis of protein may be used in studying of primary structure of a
protein and in preparation of amino acid mixtures. The latter are used as a medical
preparation for parenteral nutrition of patients who cannot use their
gastrointestinal tract (e.g. removal of large part of gut, and statement of
unconsciousness) or should not use their GIT. Parenteral nutrition is performed:
intravenously. During this procedure, the patient may get amino acid mixtures,
glucose solution, lipids (fat emulsion), and electrolytes.
PHYSICOCHEMICAL PROPERTIES OF PROTEINS
Proteins exhibit a number of physical and chemical properties which play an
important role in their functional activity. Physicochemical properties depend on
the amino acid composition of a peptide chain.
1. Solubility.It depends on several factors: oo
2) Polarity of a solvent and polarity of amino acid radicals in a polypeptide
chain. Similarities are soluble to each other.
Most proteins are hydrophilic because of polar amino acid radicals present
on the surface ‘of protein molecule. Proteins rich in polar amino acids are more .
water soluble. Proteins rich in aliphatic or aromatic amino acids are relatively
insoluble in water but are soluble in lipids. Hydrophobic side chains of such amino
acids are surrounded by lipid <molecules in biomembranes, and water-insoluble
proteins are predominantly included into cell membranes.

increases solubility of protein but
b) Temperature. The rise of temperature decreases due to its
to a certain extent, after which the solubility of protein
den i in precipitates.
| PHL ¢Ces in he pH medium can affect the charge of a protein
molecule and thus change solubility of protein.
-SkProtein olubilty is minimal at the isoelectric point (IEP, or pl). The =
the pH value at which the number of positive charges equals the numoer ©
negative charges, and overall molecule is therefore electrically neutral. This sta
of protein is called isoelectric state. At the isoelectric state, the protein net charge
of a protein is equal to zero, and the protein does not migrate in electric field.
d) Ionic strength of the solution.
Some proteins are not dissolved at disti
in the presence of very small quantities of neutral salts (I
elements). Only a few proteins may be easily dissolved in water,
smallest proteins which are highly polar, for example albumin.
At low concentrations of the salt, the solubility of a protein is proportional to
the increase of the salt concentration (salting-in), but the solubility of a protein is’
decreased by high salt concentration (salting-out).
2. Ionization.
In the water medium, proteins subject to ionization due to presence of
ionizable groups (-COOH and -NHb) at the molecule. The ionizable groups are
present at the N- and C-terminae of the protein molecule as well as at the side
chains of monoamino dicarboxylic amino acids (or acidic amino acids such as
aspartate and glutamate) and diamino monocarboxylic amino acids (or basic amino
acids, such as lysine, arginine, and histidine). Due to dissociation in water medium
at pH 7.4, the free carboxylic group (-COOH) can release (donate) proton anc
carry negative charge (-COQ), and amino group (-NHj) can accept (take up)
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proton, and therefore carry positive charge (-NH;").
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lled water but are capable to dissolve
and II groups of
these are the
Because of their side chain functional groups, all proteins become more
positively charged at acidic pH, and more negatively charged at basic pH.
Most proteins of animal origin have negative charge at physiologic pH. The
exception represents protamines and histones which are positively charged.
3. Hydration .
Dissolution of protein in water proceeds via hydration of the proteir
.... molecule. Both polar (uiicharged) and ionogenic:groups (positively and negative}:
charged) of proteins are capable to interact with water and be hydrated. Hydratior
of protein results in the formation of hydration shell on the surface of the prote
molecule. Hydration shell is composed of water molecules spatially oriented in :
definite manner around the protein. |
Water molecule is polar and has opposite charges on its two points. Becaus
of its dipolar structure, water molecule can be oriented with negative or positit:
point of the dipole to the positive or negative charge of the ionizable group ¢
protein and also to polar uncharged groups exhibiting relatively negative charg
(=NH, -C=0, -OH, etc.). Oxygen Ornitrogen atoms are electronegative and can t
weaklv attracted to hydrogen (electropositive) atom of the water dipole.

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Due to process of hydration, proteins are cap
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spontaneous precipitation, Factors. wie e factors prevent protein from
fo charge of a protein can cause precipitation of protein molecule. These
ed for separation and purification of protein molecules.
4. Amphoteric property of proteins.
The term amphoteric is used to describe the property of a molecule to have
both carboxyl group (-COO7) and amino group (-NH;"). Protein contains both
groups (-COOH) giving a molecule acidic properties and groups (-NH,) givimg
basic properties. i
5. Buffer property of proteins,
Buffers are the molecules capable to release H* or accept H” ions and thus
maintain a relatively constant hydrogen ion concentration (pH value). Proteins
contain ionizable groups that can donate or accept protons. Since protein molecules
are present in significant concentrations in living organisms, proteins are powerful
buffers.
6. Colloid property of proteins.
Proteins form colloid solutions, i.e. proteins never spontaneously precipitate
from their solutions. Proteins are able to form colloid solutions due to hydration
shell and charge which prevent protein molecule from precipitation.
7. High viscosity of protein solution,
8. Low rate of diffusion.
9. Swelling ability in water.
10. Optical activity.
11. Mobility in electric field. When current is applied to the solution of
protein, its molecules with negative charge migrate towards the anode, and those
with positive charge migrate to the cathode. This property of proteins’is used in
purification of proteins by the method of electrophoresis.
12. Low osmotic and high oncotic pressure. )
Proteins of blood bind water molecules and thus retain water confined within
the blood vessels. Due to high oncotic pressure proteins maintain a constant blood
volume in the organism. .
13. Ability to absorb UV light at 280 nm. oo |
This property is based on the presence of aromatic amino acids in proteins.
The property of tryptophan to absorb UV light at this wave-length is used for
quantitative measurement of protein concentration in solution by the method of
spectrophotometry.
14. Light-scattering preperty. }
The light-scattering property of proteins is used in modern methods of
microscopy of biological objects.

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15. “Protein molecules are incapable of passing Cin Ther is why
artificial membranes as well as across biomembranes o on the urine through
albumins and other blood proteins are not.excreted normaly
nephrons.
SHAPE: OF PROTEINS
; ular
Proteins are classified according to their shape into TWO BE lly
and fibrous. Globular proteins are compact spherical molecules y long as its ;
water soluble. In a globular protein, the length is up to 50 mes of roteins
width, and may vary from ball-like (spherical) to oval in shape. Globular p (obi
have usually dynamic functions, e.g. enzymes, transport proteins (hemosio 501
albumin), and immunoglobulins. In a fibrous protein, the length 1s ng he :
times as. long as its width. Fibrous proteins are long thread-like (or rod-1i e):
molecules that are water insoluble and usually have structural function.
MOLECULAR MASS OF PROTEINS
Molecular mass is expressed in daltons (abbreviated Da). One Dalton is.
equivalent to one-twelfth of the mass of carbon-12 (2C). ;
Proteins are high-molecular compounds, polymers containing between 30:
and 2000 amino acids residues. The mean molecular mass of an amino acid residue
is about 110 Da. Therefore, the molecular mass of most proteinbs is between 5500.
(6000) and 220 000 Da and sometimes even up to few millions. :
There are several methods most commonly employed for the determination:
of molecular mass of proteins: sedimentation analysis, gel filtration, and gel:
electrophoresis.
&
1). Sedimentation analysis or yltracentrifugation. : 5 :
The technique was developedfSwedish biochemist Th. Svedberg (Noble
Prize Laureate, 1926). The construction of ultracentrifuge allows to get the rate of:
sedimentation of large molecules up to 100 000g and more (this means that
centrifugal acceleration is 100 000 times as much as Earth’s gravitation). Thet
ultracentrifuge is equipped with a special optical device to register sedimentationt :
of protein molecule when it migrates down the height of the ultracentrifuge cell ok
its bottom. As the protein particle migrate, a sharply defined solvent-protein:
interface is formed, whose boundary line
is recorded automatically to determine
sedimentation rate. Sedimentation rate is expressed by sedimentation constant The:
sedimentation constant is expressed in seconds. The sedimentation constant 3 5
value 1- 107 second is a unit named Svedberg, S. Then the sedimentation c a.
and other parameters are used for calculation
of molecular mass
of a onstant:
edn sedimentation analysis is a laborious, costly and time-co
2). Gel filtration, or gel chromatography, or molecular sedi
For this procedure, Sephadex-containing columns are used Sephdex i i
. X is a
polymer forming granules (beads) with pores or cavities of a particular size. Large:
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protein.-
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proteins cannot enter the cavities, and so rapidly pass through the column, around
the granules. Small proteins diffuse in and out the pores, i.e. small proteins can
enter the cavities, then come out and re-enter into another pores. Thus, small
proteins migrate through the column more slowly and are retained in the column
longer. As a result, in this method large proteins will come out from the column
first, or sooner than small ones. The smaller is molecular mass of a protein the
slower it moves. The method acts as a molecular sieve: due to the difference in
rates of protein migration through the column, the protein mixture may be
separated into divided sma bands which are then collected ately.
In molecular sieving, one should start with calls on of the Sephadex
column, For this purpose, a mixture of proteins with known net molecular masses
1s allowed to pass through the column, and the elution volume is measured.
Elution volume is a volume in which a protein has completely left the column. The
graph is obtained by plotting the logarithm of the molecular masses of known
proteins against the corresponding elution volumes. Then the unknown protein is
passed through the same column and the molecular mass is calculated from the
measurement of the elution volume of this protein, as compared with the standards.
A variant of this method is thin-layer gel chromatography. The length of
run (in mm) to which a protein migrates through a thin Sephadex layer is
logarithmically related to the molecular mass of the protein.
3). Gel electrophoresis.
First, standard proteins with known molecular masses are separated by
electrophoresis. Protein mobility is measured, and a calibration curve relating the
logarithm of the molecular mass to protein mobility is plotted respectively. Then,
the unknown protein is also subject to electrophoresis under similar condition, the
mobility of unknown protein is measured, and the molecular mass is determined by
comparing with the graph.
PRECIPITATION REACTIONS OF PROTEINS
Protein solutions are very unstable and proteins may undergo precipitation
under the action of cértain factors. Precipitation reactions of proteins may be
divided into two groups: reversible and irreversible reactions.
Reversible precipitation of proteins
Reversible precipitation is so named because functions of a protein
precipitated are not impaired, and after removal of the agent that had caused
precipitation the protein may be dissolved and exhibit all its properties and
functions.
The charge and hydration shell of a protein molecule are responsible for
the stability of protein in a solution. The agents which neutralize the charge and
remove hydration shell cause precipitation of proteins.
Methods for reversible precipitation of proteins

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1. Cohn’s method. In this method, aqueous ig act a otatei
temperature (-3 to -5°C) are used for protein precipitation. Ethanol
as a dehydration agent which removes hydration shell. : initation i
2. Saltingont This procedure is commonly used for protein prec a
clinical practice for analysis of proteins in the blood serum and other orc ary
fluids. Also salting-out is used in preparative enzym ology for preliminary
precipitation of ballast proteins or for separation of certain enzyme.
or . ich
Salting-out is a reversible precipitation of proteins when a neutral salt at hig
concentration is added to a protein solution. Usually, for this procedure salts of
metals belonged to I and II groups of elements are taken, more often NaCl (so um
chloride), Na,SO, (sodium sulphate) and ammonium sulphate, (NH) 250s. tho
result, the hydration shell is removed, a charge of the protein 1s neutralized, and the
protein is precipitated.
:
~ A number of both protein and salt characteristics (factors) may influence the
efficiency and velocity of salting-out.
Protein factors that influence the velocity of salting-out |
1) The dimension of net charge of protein molecule (the more is the net:
charge the slower is salting-out).
2) The dimension of hydration shell on the protein molecule (the more is:
the hydration shell the slower is salting-out). 2
3) Shape of protein molecule (globular, ball-shaped, or spherical, proteins:
are easier to precipitate by salting-out, and vice versa, fibrous and ellipsoid-shaped:
proteins have more buoyancy (floatage). :
4) Molecular mass of a protein (the larger is the protein the easier it is
precipitated). ¥
Salt factors that influence the velocity of salting-out
1) Concentration of the salt used. As a general
molecular mass of a protein the less is the amount of salt requ; ipitati
. oo a red ;
Ammonium sulphate is widely applied in the clinic laboratory hy a
globulins and albumins in the blood serum. As proteins differ by thei onl er i.
weight, charge, hydrophilicity, etc., the different concentrations of the same sal
salt
are required for the salting-out of different rotej
A R 11S. s « .
molecular mass respecting to albumins) are precipitate] J PS (with higher
saturation) of ammonium sulphate. Albuming which are o saturation (50
are precipitated at full saturation (100 % saturati .
2) Nature of the salt. The precipitating hil ammonium sulphate.
rule, the higher is the
Ca**>Li*>Na* > x 2 nl + ONS ~

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Sodium chloride exhibits lower
suiphate (according to the position of ions
“weaker” 1s the salt the higher concentratio
27 the protein. If the salt is “strong”
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precipitating ability than ammonium
in the Hoffmeister’s Tow); therefore the
n of the salt is required for Precipitation
e pro it is efficient at lower concentration to cause
=7=cipitation of protein. E.g., globulins may be precipitated by either ammonium
suiphate or sodium chloride. The former salt is “strong”, the latter one is “weak”.
Therefore, globulins may be precipitated at half saturation of ammonium sulphate,
2ut for the same effect, full saturation of sodium chloride should be taken.
Normally, reversible precipitation exerts mild effect on a protein; the
orecipitated protein may be re-dissolved in water after removal of the salt
precipitators) by dialysis, or gel chromatography, or dilution, and the protein
regains its native biological properties and functions. .
Irreversible precipitation of proteins
(Denaturation of proteins)
Denaturation is a destruction of unique native structure of ‘protein molecule
with the resultant loss of its biological activity, i.e. .loss of functional and
physicochemical properties. Denaturation of -proteins is sometimes named
, ureversible precipitation, but this is not quite Tight because sometimes being
denatured the protein may remain still soluble and may be precipitated by bringing
10 isoelectric pH. :
There is a variety of physical and chemical factors that cause denaturation of
protein.
Physical factors:
1. Heat. Most proteins are denatured as their solutions are heated to above
30-60° C. But proteins can preserve their biological activity for days when the
solution is kept at low temperature. E.g. alkaline phosphatase in blood will be
inactivated within two days when kept at 20° C; but will be active for seven days at
4° C, 20 days at -20 ° C and for three months at -80° C. Proteins can be preserved
for years when lyophilized. Lyophilization (freeze drying or drying in vacuo by
water sublimation in a’frozen state) is the process by which water is evaporated at
very low temperature in vacuum.
Native proteins are often resistant to proteolytic enzymes of gastrointestinal
tract, but denatured proteins have more exposed sites for enzyme splitting of
proteins. Cooking leads to denaturation of proteins, and cooked foods are more
ily digested. :
i ; : Radiation of different types (0+, §-, - radiation, X-rays, ultraviolet rays).
3. High pressure, hho ; a
4. Vigorous shaking or intensive stirring of a protein solution.
Chemical agents. iy
1. Inorganic acids (HCl, H;SO,, HNO;). Phosphoric acid does not cause
precipitation of protein.
2. Organic acids (trichloroacetic acid, sulphosalicylic acid, acetic acid).

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3. Inorganic basics [NaOH, KOH, Ba(OH), Zn(OHDa).
4. Organic basics or so called alkaloid reagents (OTP e 4. or silver, mercury;
5. Heavy metal salts (salts of Ag, Hg, Cu, Pb, . ’ dium of the body
copper, lead, iron, zinc cadmium, respectively). In alkalin © Salts of heavy metals
(pH 7,34) proteins have net negative charge, or are rt in molecules and
contain positively charged metal ions that can complex with prote white or milk
lead to precipitation of the protein. Based on this principle, raW es incidentally
is used per os as an antidote for poisoning if heavy metal salts Were ’
swallowed,
nds and
Denaturation is accompanied by the rupture of non-covalent bon
disulphide bridges (i.e. non-specific alterations in secondary, tertiary and
quaternary structures of protein molecules), while the peptide bonds Fema
unaffected (i.e. primary structure is not altered during the process of denaturation).
The globules of native protein molecules uncoil to form random and disorder ed
structures.
Thus, denaturation leads to numerous changes in the protein structur
physicochemical properties:
- unfolding of polypeptide chain;
- loss of secondary, tertiary, and quaternary (but not primary) structures;
- rupture of all non-covalent bonds and disulphide bridges (i.e. all internal
bonds that stabilize the native structure of proteins); but peptide bonds
remain unaltered;
- increase in the number of free HS-groups;
- increased viscosity of protein solutions;
- loss of solubility of proteins.
e and
All these changes lead to the loss of biological activity (functions) of proteins, Pe.
catalytic, antigenic, or hormonal functions, etc.
If the action
of the agent causing denaturation is of short duration and can be
promptly removed or ceased, the phenomenon of renaturation is possible with a
complete testoration of initial structure and native properties of the protein
including its biological activity. But many proteins (e.g. albumins) could not be =
. natured by the removing of the physical agent that has caused denaturation.
Use of denaturation in medical practice
1) Precipitation of proteins with HNO; or sulphosalicylic acid i )
as a test to detect the presence of protein in the urine. yhie acid is carried out
2) In clinical laboratory trichloroacetic acid is
ballast proteins from blood and other body fluids. Sad
3) Sterilization of surgical instrument : ;
operation, | HE and surgeon’s hands before the
4) Conservation (preservation) of food products
5) Fixation of tissues by formalin (aqueous solution of formaldehyde)
COLOUR REACTIONS OF AMINO ACIDS AND PROTEINS
precipitation of
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Colour reactions are designed to detect the
in a solution. Colour reactions are divided into
and specific reactions. Common reactions are biuret reaction (for all the proteins)
and ninhydrin reaction (for all amino acids
i : and proteins). Specific reactions
(xanthoproteic reaction and Fohl reaction) are }
: used to detect only certain amino
acid bo a protein molecule and in a solution of amino acids.
Biuret reaction®
Cupric uSQOq) in an alkaline medium
|
(NaOH)
form a complex of
violet colour with peptide bond nitrogen. The rea
J L ction is carried out at room
emperature.
The test is positive if minimum two peptide bonds are
available
in a~
peptide; individual amino acids and dipeptides (dipeptide contains one peptide
bond) will not answer this test. The name of the reaction is derived from the
compound biuret (bi-urea), NH;-CO-NH-CO-NH,, which is a condensation
product of two urea molecules (NH,-CO-NH,), and also gives a positive colour
test. ®
presence of protein or amino acid
two groups: common (universal)
Biuret reaction is used for quantitative estimation (measurement) of protein
in a solution.
Ninhydrin reaction.
All camino acids when heated with ninhydrin can om purple (violetblue)
complex called Ruhemann’s complex. The reaction is carried out a ing. The
“ninhydrin reaction is applied for both qualitative and quantitative estimation of
amino acids. It is often used for detection (identification) of amino acids in
chromatography.
Xanthoproteic reaction (Moulder reaction).
Amina acids containing aromatic ring (phenylalanine, tyrosine, and
trypfophan) can undergo nitration with concentrated nitric acid (HNOs) when
heated. The end product is yellow in colour which is intensified in strong alkaline
—
medium. .
Fohl reaction (sulphur test for cysteine).
i steine containing proteins are hoiled with strong alkali,
ic sulphur splits apd-forms
sodium sulphide. The latter, on addition of lead
oat Trienaive heating produces lead sulphide as a black precipitate.
" Methionine does not answer this test because sulphur in methionine is in the thio-
ester linkage which is difficultto break.
EINS
R THE QUANTITATIVE MEASUREMENT OF PROTE
METHODS FO ? IN A SOLUTION :
The methods that are usually used in clinic laboratory for quantitative
estimation of protein in a solution (or biological fluids) are divided into: 1)
colorimetric method, and 2) spectrophotometric method.
1. Colorimetric method. TS——
The intensity of colour produced by biuret reaction is estmatec.

a Q4) in 20°
Principle of the method. Cupric 1003 (CuSQ protein
(NaOH) form a complex with peptide bond nIfro8®
producing violet colour. The intensity ©
of peptide bonds and protein concentration
one-step process, and is the most widely use€
estimation in clinic laboratory.
This method is used in clinic laboratory for
protein. i
Clinical application. Normal concentration of total po
g/L. Total serum protein may be separated into albumins an g
are divided into several groups: o-, 0-, 8- and globulins. tein is named
Hyperproteinemia. The increase of total serum prote
hyperproteinemia. Causes of hyperproteinemia: a
FY Hyperproteinemia due o ¢the increase of both albumins and globulin? os
the blood serum (relative hyperproteinemia) takes place In dehy ra!
(water loss) of the body. Dehydration in turn may be caused by a
pathological sweating (fever) or urination, profuse diarrhea and repeate
measurement of total serum
m protein is 65-85
bulins. Globulins
vomit. .
2) Hyperproteinemia due to the increase of globulins takes place In
plasmocytoma (myeloma disease, or multiply myeloma), collagen diseases,
and in chronic infections.
Hypoproteinemia. The decrease of total serum protein is named:
hypoproteinemia. Causes of hypoproteinemia: :
1) Hypoproteinemia due to the decrease of both albumins and globulins
in the blood serum takes place in cancer cachexia (emaciation of the body:
because of cancerous process), malnutrition and malabsorption. i
2) Hypoproteinemia due to the decrease of albumin only in the blood;
serum may be caused by: :
a) the decreased synthesis of albumin in the liver (hepatitis, cirrhosis i
or i
b) the increased excretion (loss) of albumin in the urine
syndrome, chronic nephritis).
2. Spectrophotometric method.
Proteins absorb ultraviolet light at 280 nm. This is due t
tryptophan residues in the protein molecules. The absorption
test solution is measured by spectrophotometer and
protein concentration in the solution.
A spectrophotometer has all the bag; |
colorimeter with more sophistication. The light IW Sr of photoelectric’
converted into electrical impulses and finally passes i ough the cuvette is then
The advantage of the spectrophotometer re the display unit.
former is 1000 times more sensitive, E.g, protein in
may be tested by colorimeter. If the prog; solut
then colorimeter is ineffective, and only sp
spectrophotometer is much more costly th
(nephrotic
0 the tyrosine and
of UV light by the:
the value is proportional to
ectrophotometer
an a colorimeter,

[TELE
A
STRUCTURE OF PROTEINS
Protein molecule is a product of polymerization of 20 different amino acids
(monomeric units) bound to each other not randomly but in a strict correspondence
with the genetic code.
Proteins have different levels of structural organization, i.e. primary,
secondary, tertiary, and quaternary structure.
Primary structure *
Primary structure is the sequence of amino acids bound to each other with
__ peptide bonds with resultant formation
a polypeptide chain. The repeating unit of a
polypeptide chain contains three sequential bonds: N-C,, Co-C and C-N bond. The
C-N bond is peptide bond. Primary structure is determined by genetic information
encoded in DNA.
(EE A
eden IS NEP I
| | | | | |
0 R H 0 R 0
(A
NE AN
CN N—C, C,~—C
Peptide
bond
Fig. Fragment of polypeptide chain.
Characteristics of peptide bond
"1. The peptide bond is a partial double bond. The distance between C and N
atoms is midway between single bond and double bond.
2. The peptide bond is rigid bonds: because of its partial double-bond character,
the peptide bond has no freedom
of rotation. The side chains are free to rotate on
either side. Also rotation is permitted about the N-C, and the CoC bonds. The
peptide bond limits the rate (intensity) of spiralization of a polypeptide Chain.
3. The peptide bond is planar, i.e. the carbon atom (with oxygen) and nitrogen
atom (with hydrogen) of a peptide bond together with CH-groups adjacent to the
peptide group lie in single plane.
4. The C-N bond is in rans configuration, i.e. oxygen atom of the carbonyl group
and the hydrogen atom
of the amide nitrogen are on opposite sides of the peptide
bond.

y Lo Me 9 y SN 0 .
& H ye 7 A I i ~~ = C—
= | HC ui: —N-—CH—C— |
wr TNTRTETISUL AN Lo
ns RN s GIN 0 SOR
_/ RY i, ANB rd
x CN NC. CC
VY Peptid
Na 7 od
PA 7 ¢ of the peptide bond. The
J rs Fig. Properties of a peptide bond. Planar characte
ba JX peptide bond"is not free to rotate. The N-Cq and C.-C bo
-N~ bonds in proteins are in trans configuration.
nds-can rotate. All peptide
Role of primary structure
> 2¥ 1. Primary structure determines all the higher levels of protein organization ;
> 2Y 7 (ie. secondary, tertiary, and quaternary structures are dependent on the °
OIA
~ _ 3¢ ° primary structure). :
& ra 2. The linear sequence of amino acid residues in a polypeptide chain °
4 determines the three dimensional confignration of a protein, and the structure of : |
a protein determines its functions. The side-chain radicals define “the spatial ‘
© structure and functional diversity of protein molecules. Z
Ed ¢ 3. The amino acid composition of a polypeptide chain determines -
physicochemical properties of a protein. Proteins rich in aliphatic or aromatic
Sy amino acids are relatively insoluble in water and more soluble in ce|] membranes :
TL NY Proteins rich in polar amino acids are more water soluble. :
EN 4. Each protein has a unique (specific) primary styy . g
< the unique fi ne ion_thij stein perfor Even a si eture responsible for :
5 * single ami . E
S Ino acid change i
“(mutation) in a linear sequence may lead to alteration ; : :
properties and to loss of biological functions of the protein, nn physicochemical
} E.g., sickle cell anemia. In this disease, a person
Normally, human adult hemoglobin (Hb A) is data oe tan hemoglobin.
and two identical 8 chains. Sickle cell anemia results from 5 «; entical « chains
substitution in the 8 chain of hemoglobin: the * amino ey amino acid
(normally this is hydrophilic glutamic acid) is replaced by hyd 1 m the B chain
valine. This single substitution leads to the formation of 1 rophobic amino acid
This abnormal HS, on the release of oxygen in tissues Ca S (Hb S). §
and is polymerized to form spindle-like (rod-like) crystals in Hg poorly soluble
Due to this, the erythrocytes assume sickle shape, beco Side red blood cells.
normal ones, and undergo hemolysis which |eaqs to ceve Me more fragile than
also characterized by painful vasoocclusive crises: in capil: hes disease is
» the sickle cells
“aR
PRA
S
ei
a
d
ibis

N
:
INT
L
i= small plugs which result in the intermittent clogging of capillaries and
scruciating pain. Occlusion of major vessels can lead to infarction in organs.
5. Primary structure determines both individual and species specificity of
cooteins. Any human individual has some proteins with a mino acid sequence
<=erent from that of the same proteins .of another individual. This discrepancy in
#720 acid sequence should be taken into account on blood transfusion or
—zasplantation of organs.
Secondary structure
Secondary structure is a configuration of a polypeptide chain, i.e. the way
= polypeptide chain is folded, or packed. Secondary structure is formed not
==2omly but in a strict accordance with the linear sequence of amino acids in the
FTEmany structure of a protein.
There are several types of secondary structure: the orhelix, the B-
. “&mlormation (B-pleated sheet), the collagen helix, etc.
The o-helix. —
The o-helix is the simplest arrangement the polypeptide chain could assume
#7 Its rigid (incapable for rotation) peptide bonds. Also, this is the most common
“z= stable conformation for a polypeptide chain. The o-helix is a rod-like spiral
structure. It is formed when the polypeptide chain is wound around an imaginary
xs into a coiled spring-like conformation, The side chains of amino acid
-—
residues are oriented to the outside of the helix. Thus, the polypeptide chain forms
== backbone, and the side chains are not involved in the formation of secondary
sTacture.,
The o-helix winds in a right-handed manner, i.e. turns in a clockwise fashion
zreund the axis.
The repeating unit of the a-helix is a single turn of a helix. Each single turn
:2z2 pitch of the spiral) occupies about 0.54 nm along the axis and includes 3.6
zmino acid residues per turn. A stretch of one amino acid (the distance between
~ 2djacent amino acids) is 0.15 nm. The structural configuration of the polypeptide
huey
_%82in is repeated each five tums and includes 18 amino acids,
| SR Fig. orHelical structure.
> 015m (14 The pitch (the distance between
~~) oT reste. correspondent points per turn) is
A 5 Sie 0.54 nm and contains 3.6 amino
a & (3Bresidues) acid residues (0.15 nm per 1
amino acid). The helical repeat is
3% m 2.7 nm and includes 18 amino
18residues

=
(45 W pleated sheet) is
- The d riving force in the building up of o-helix (as W fri LP os bonds. —
"the ability of CO and NH groups of peptide bonds to — +d
: from NH group 0
Hydrogen bonds are formed between BE ated four residues further along
and the carbonyl oxygen of amino acid th a. : hydrogen bond
the chain. Alena ied peptide bond in the a-helix participates in hy
formation, except for peptide bonds close to each end of {hi hell. ain) hydrogen
Thus, a-helical structure is stabilized by internal (in :
bonds. Fw ad
Hydrogen bonds are non-covalent bonds and are rather weak oo ies
since the nuriiber of hydrogen bonds in the protein molecule " oh ontains 3.6
provide for twisting polypeptide chain into a helical structure which ¢ es and
amino acid residues per turn and gives the entire helical structure compac n
The stability of o-helix may be also partially maintained by disulfide bonds
a eT vi y . p H ¢ Ix
Certain amino acids do not allow forming of o-helix. Eg, helix Bets
broken by proline residue which has no -NH group available forthe formation of - ¢
. Dt “sail H 1 1 ix :
intrachain hydrogen bonds. Thus, proline forms a kink in an o-helix .Also,
E.g., in regions with large number of adjacent lysine and arginine residues, which =
‘have the positively charged R groups, the side chain radicals will repel each other
and prevent formation of chelix. For the same reason, regions with numerous - _.
aspartate and glutamate residues, which have negatively charged R groups, are also
incompatible with o-helical structure. Amino acids that have large, bulky side
chain (asparagine, tryptophan, serine, and isoleucine) can also destabilize an o-
helix if they are close together in the chain.
Glycine destabilizes crhelix
too. This amino acid has the lest side chain R ©
(only H) and this gives more flexibility to polypeptide chain. .
Each protein is characterized by the percentage of a-helical structure Ge
helicacy, of its polypeptide chain. Keratins that are present in hair, skin ’ adil.
horn, hoof, etc., have the typical (100 %) orhelical structure. Many lobul a
proteins contain c-helical regions combined with linear ones afd 8 Jeated
sheets. Globular proteins often contain several types of secondary bend hh
same polypeptide chain. ; > st in the
-Pleated sheets (B-conformation or SB-structu re).
In the B-conformation, polypeptide chain is al " :
zigzag structure. In B-structure, the distance btween py ws forms
single polypeptide chain is:0.35 nm (comparing t0 0.15 nm in ochelica - sy :
In B-pleated sheets, one or more zigzag polypeptide cham og lica structure). :
side to form a parallel structure resembling a series of p] Ee side by |
arrangement is called -pleated sheet. Ot pleats; therefore this
The (3-sheet structure is stabilized by interchai
formed between carbonyl (C=0) group of a pa re of :
a i —NH group of

-2ntide bond in the neighbouring polypeptide chain. The R groups of adjacent
=ino acids protrude from the zigzag structure in opposite directions.
There are two types of 3-pleated sheets: parallel and antiparallel. In paralle|
-pleated sheet structures, adjacent polypeptide chains have the same N-terminal to
C-terminal orientation. And in antiparallel 8-sheets the N-to-C terminal orientation
of adjacent chains is arranged in opposite direction. The repeat period in a §-
o.zated sheet is 0.65 nm for parallel and 0.70 nm for antiparallel conformation
comparing to 0.54 nm in o-helical structure).
“—10.650m —»
(0.70 nm)
Fig, Zigzag structure of B-pleated sheet and its repeat period.
Fibroin, the protein of silk or spider web, has the polypeptide chains
predominantly arranged into 3-conformation.
N-terminus C-terminus
I
mL Jets PAN ZN aN Y NT / + N-terminus
nus N ¢ C HY ke NOH
R Oo
N-terminus C-terminus
Fig. Antiparallel §-pleated sheets with interchain hydrogen bondsr

TL
tide
le polypeP
1 of a sl g . chainl
) g-Pleated sheets may be formed by Be he: B- heets 11 ich the itself.
chain. Also, globular proteins usually a oe carn and fol os
: . ‘ Cc
N
abruptly reverses direction, 1.e. is +1 the surfac feo ot P cither 0
;

X
Glycine and proline often occur It 2 cture di n
ee are also natural proteins that have epresentative of suc
helical or B-pleated sheet structure.
Pod
collagen, a fibrous protein which 18 the 1
major fibrous element of connectiv® tis ; : handed hel
.
]
e
(about 30 %). Individual collagen po ypep (in ob eli
slightly extended and contain 3 amino acid residues Per turn (
acid residues make one turn). . i
VER
RYE
SY
i
y
Fig. Secondary structure of collagen. Left-handed helices are extended
and contain 3.3 amino acid residues per turn.
PTE
TER
Tor
a
{im
ts
Tertiary structure
conformation of a protein is Tied a its tertiary structure. This structure reflects +
the overall shape of the molecule, the manner in which a polypeptide chain is 5
ed
*o
folded, and spatial arrangement of the secondary structure within a confined :
The three-dimensional (or spatial), folded and biologically active :
space.
“properties: 1) they are water insoluble, 2) thread-like (rod-like) molecules. 3
. ———
body, i.e. their function is to provide support shape anc a =
—
) and ex x : 5
re pe and external protection to the «
whi / Nie radi x with ery
while Ieydeophilic radicals are on the surface of iki aqueous surrounding §
interact with water. The presence of amino acids th In molecule where thevs.
polvpeptide chain ( mainly such as proline and aly at form kink, bend or turn of =.
e
g.
Scanned by Gamscanner =

Yhich differznt segments of a polypeptide chain can fold back on each other
o folding of a polypeptide chain, the amino acid residues thar are locates
considerable distance from each other in the primary structure may come 1o
proximity and interact with each other within the completely folded swuctur
protein to produce bonds. stabilizing tertiary structure. This folding gener
compact form of protein that acquires thermodynamically favo
conformation. In such a state, the protein molecule assumes stabilit
corresponds to minimum of free energy.
he The three-dimensional tertiary structure of a protein is stabilized by di
types of bonds: hydrophobic, electrostatic, hydrogen bonds (all of whi
non-covalent) and also by covalent disulfide bond.
Hydrophobic bonds. They are formed by interaction between no
hydrophobic side chains of alanine, valine, leucine, isoleucine, methiont:
phenylalanine.
-= Electrostatic bonds (ionic bonds). They are formed between Two ¢
charges. Positive charges are produced by e-amino group of lysine, guan
group of arginine, and imidazolium group of histidine. Negative “cher
produced by f3- and y-carboxy! groups of aspartic and glutamic. acids. }
Hydrogen bonds. They are formed between electronegative ¢
(oxygen and nitrogen) and positively charged hydrogen atoms. Typical
bonds occur in a protein molecule between:
a) two hydroxyl groups (-OH),
b) hydroxyl group (-OH) and carbonylgroup (-C=0) of a peptide be
¢) hydroxyl group (-OH) and ionized carboxyl group (-COQ7),
d) between —C=0 and -NH groups of polypeptide chains:
=
8) RCO HAO—(-R 9 HGR RC
| L oy
%
# NH vw O=
b) id rd
R CHR RCH
CHAO—H we D=ics N
NH
c) 0
R~C_ |
Ow H=0- _S-R
Fig. Typical hydrogen bonds occurring in protein molecules.
®

-. : -egidues. ;
ge % rq cysteine residu .
. :Disulfide bonds are formed between two CY er the termination of protetl
The build up of tertiary structure oe ~ ary structure. The
svnthesi : put is define pa oe
sathes s and occurs spontaneously eptide chain (i.e. amino a
.« information. 101
A as genetic infor : ole
linear one-dimensional structure of a poly
or primary structure) is encoded in the DNA ich determines
acid sequence contains conformational information os e, OF
formation of a protein molecule of definite shape. Thus, he tert” cid
spatial arrangement of a protein molecule is predetermined by : ino acid
sequence
of polypeptide chain, or by the size, shape, and polarity 01 2m
side-chain radicals. - ical
Tertiary structure is a native conformation of a protein: all the biological
functions of proteins (catalytic, antigenic, hormonal, and others) are exhibited bs
the tertiary structure. Only tertiary structure of a protein is able to exhibit
biological functions. )
The shape or conformation of proteins can be altered by interaction with
other molecules, called ligands. Because of this ability to modify the form,
proteins seem to be alive or dynamic molecules, responding to the ever-changing
environment within cells, blood, or extracellular fluid.
Large globular proteins (i.e. those with more than 200 amino acid residues)
often contain several compact units called domains. Domains are structurally
independent segments that often have specific functions. The domains are usually
connected with relatively flexible areas of protein.
Fig. Domain structure of a protein.
Tertiary structure of fibrous proteins is adapted for structural function. E
a-keratin, fibroin and collagen have fibrous tertiary structure. Sa
a-Keratin is present in hair, wool, skin, horns, fingernails, feather. ot
Secondary structure of o-keratin is o-helix. Its tertiary structure is I; ne
three strands of o-keratin secondary structures are wrapped about dor Ne
produce a supertwisted (supercoiled) left-handed structure calle eet Oiler To
supertwisting amplifies the strength of the overall structure
twisted to make a strong rope. d
! d protofibril. The
Just as strands are
Fig. Tertiary structure of a-keratin.

»
&
This swucture is held together by hydrogen bonds and also is strengthened
by covalent disulfide bonds. Eleven protofibrils are packed together to form
microfibrils (quaternary structure of o-keratin). Hundreds of microfibrils are
packed together to form a macrofibril. Each hair contains several macrofibrils.
Collagen. Collagen is the most abundant protein in vertebrates. It is present
n bones, cartilage, tendon, and connective tissue. Collagen has a unique secondary
structure distinct from a-helix. It is left-handed and contains 3 amino acid residues
per tum. These structural constraints are unique to the collagen helix and are based
on the unusual amino acid content of collagen. Typically collagen contains about
35 % glycine (one-third of the amino acid residues), 11 % alanine, and 21 %
proline and hydroxyproline. The amino acid sequence in collagen is generally a
repeating tripeptide unit, Gly-X-Y, where Gly is glycine, X is often proline, and Y
is often hydroxyproline. Hydroxylysine is also found in the Y position
In tertiary structure, three separate polypeptide chains are supertwisted about
each other to form aright-handed triple helix (superhelix) called tropocollagen
Fig. Triple helix of collagen molecule.
The high proline content of collagen explains for its lack of o-helix. Also.
the proline and hydroxyproline residues allow the collagen helices to be closzly
twisted and packed. Besides, only glycine residues have a small R group for the
space available between the tightly twisted individual collagen helical chains. This
packing of polypeptide chains gives stability to the triple helix and provides
rupture strength greater than that of a steal wire of equal cross section.
Molecules of topocollagen are packed together to form a collagen fibril
(quaternary structure). In collagen fibrils, tropocollagen molecules are aligned in z
staggered fashion and are cross-linked by unique covalent bonds between lvsine
and hydroxylysine residues to enhance the strength of this protein
rmie—et wast ¢ roc if a
£ ra
va ya u
/ /
Aged: « DP RERENSSS GESTED
celfege Section of calla en’
me 18&u jes mel€cule -
Fig. Structure of collagen fibrils.

- - ; eptide
: : torweb. Its polyP
Fibroin: This protein is present in silk and spider g-pleated sheets
srzins are in B-configuration and are arré eo and glycine i
t==izrv structure). Fibroin is rich in alaninl ashitie _grvs
-=zmvely small R groups. This allows close PE = tabilized bY interchain
zmomass 10 the protein Tertiary structure of f1be wi sheet, thus
~virogen bonds between all peptide linkages of each
ce io: 5 ca
“=xiniline of the structure. Silk does not stretch be ~ NAVA 5
. .
AN - . - Ya " A
= ~zz¢v highly extended. oo Woe, VOR «
fracture 2 Se vn nT a et
Quaternary s jp NTN ~.
~ a)
Most proteins with high molecular weight (larger than 50,000-100.000 D2.
consist of more than one polypeptide chain. Quaternary structure 1S a co foeular
wa more separate polypeptide chains which are assembled to form sup! amolect
multisubunit structure. Lu . i»
A multisubunit protein is called multimer. Multimeric proteins can have
fom two to hundreds of subunits. Each polypeptide chain of multimeric protein is
named a subunit or protomer. A multimer with just a few subunits is called
oligomer. Depending on the number of protomers, the proteins are called as
dimmer (2), tetramer (4), etc. Multimer protein may be composed of identical or
non-identical subunits. Most multimers have identical subunits or repeating groups
protomers.- Each separate protomer exhibit no biological activity. After removal
of the factor caused dissociation of protomers, the separate subunits exhibit ability
of self-assembling: they re-associate (re-unite) to restore the initial N
structure and functions of the multimeric protein. quaternary
Hemoglobin is the first protein for which the quaternarv sp
determined. Hemoglobin contains 4 polypeptide chains Ge . structure was
tetramer), and four heme prosthetic groups. The protein . g emoglobin is a
consists of two identical o-chains and two identical B-chain., rion, called globin.
a and @ are not the types of secondary structures, je. poy S (note that In this case
sheets, but names of polypeptide chains). : a -helix and B-pleated
Proteins which have quaternary structure mg 34g
cooperativity between protomers. If the small Pid exhibit phenomenon of
to one protomer, this facilitates the binding of Jjgap (ligand or effector) binds
when the first O, binds 10 a molecule of hemo = ‘ N
; €n the first 0O- binds.
es he binding of the fourth

#3 Chirvd_iY
4 =
[ETHODS FOR SEPARATION AND PURIFICATION OF PROTEINS
-
There are thousand of dirfereat types of proteins in cells. Separation and
] surification of proteins is jpEcessary’ 10 get pure protein in order to study and
.ndeystand iis functionand propertizs. The source of proteins from humans and
znimals is commonly blood and tissues. Sometimes microbial cells (bacteria),
] 2iznis and veast may be us wa.
Prossanre sequence for purification and separation of proteins includes three
major sep
| 1) homagsnization ofbiological material;
2) exuaction (solubilization) of proteins;
3) fractionation. i.e, purification of proteins. +
3 Homogenization of biolo. ical material. Prior 10 isolation of proteins from
© owological material, it is necess v 10 disrupt, break, or disintegrate cells or tissues
© by grinding them to a homogenous state. The procedure is called homogenization.
.. 11 1s carried out in isotonic solutions at physiologic pH and at 4° C 10 minimize
prowin denaturation during this procedure. A number of different techniques 1s
152d Tor homogenization: special homogenizers, ball mills, ultrasound (cell walls
re broken by ultrasound vibration), multifold freezing-melting (the freeze-thaw
technique) extrusion method (squeezing of a frozen biomaterial through a pin-hole
Ther under pressure), “nitrogen bomb’ method by which the cells are init zilyv
zllowed 10 be saturated with niwrogen under high pressure; then the pressure is
made decreased and abrupt formation of gaseous nitrogen makes the cells
“explode™).
~ Extraction of proteins. It is usually combined with homogenization. For the
sahvation and ue extraction of proteins from biological material, different
1sQzonic solutionsare used, e.g. such as buffers (phosphate, citrate, borate butter
ris-bu ffer, etc.), 0 “0 saline solutions, organic solvents (sucrose solutions,
zcueous glycerol solutions), and also. detergents) The latter are used 10 ceswoy
a obic protein-iipid and protein-protein interactions and thus release prow
(enzy es) tightly bound to membranes.
Fractionation of proteins. Due 10 previous steps, proteins from vielagical
wi
© 5
a on
peferent+ charge of proteins, thair different size, binding 1properties, and solubili "n
The methods used for fractionation of proteins include:
- selung out,
- chromatograniy,
- electrophoresis,
- ulracentrifugation,
- immune methods (imnischemical technique). — bo otiincs ire
ol
Salting out. The solubility of a protein depends on the concentration of
cissolved salts. only 2
2 TenWW proteins may be easily dissolved in water, i.e. only th
smallest and highh
I proteins, e.g. such as albumin. Most of proteins can be

w) wp LE ALR
~
cooper ative binding makes hemoglobin especially efficient at fulfilling its role as
z ransporter of O:. .
~~ Many enzymes (proteins with catalytic function) have quaternary structure
zmong them fauy acid synthetase, lactate dehydrogenase, regulatory enzymes
protein kinase
~
and phosphorylase. Many structural proteins (fibrous proteins) have
zlso quaternary structure, 2.g. keratin, fibroin, collagen.
- va bl
The occurrence of quaternary structure has certain advantages over proteins
nat can exist only in tert ary suucture.
1. The thrifty use ofgenetic material.
Much shorter fragment of nucleic acid is needed for coding of much smaller
poivpeptides, and this nucleic acid can be efficiently used over and ov Yr QC 5a 1
er agalr 10
make ma
all
all
ny copiss of the subunit, It is simply more efficient to meke many copies
poly peptic ¢chain than one copy of a very large protein:
2. The existence of quaternary structure reduces the error frequency du
ot asm
co? OL I” ing
protein plosynthsst s.
The error frequency is about 1 mistake per 10,000 amino acid residues. Buz
n this low rateresus in a high probability ofa damaged protein if the protein is
wio fates The a for incorporation of a “wrong”
tor oro nrg
lgrgs proteinarthan. for a small one.
amino acid in a protein
is greater for
ow
Lo
—
jo]
—
Ww
ag]
IN]
8
» Qe logical function orf ne
protein by wav or 2ss ocizrisrdissacistion of subunits.
1. For the cali, mansport of smaller subunits from the site of their
Io she sité of theif D
their functioning 1s more advantageous (energy-efficien
protein. 4
e
5. In supramolecular complexes, replacement of smaller or damaged
components can be fulfilled more effectively.
9

-
" H
> J S
ncentret 0 (
dissolved’ in water only if salt is added at a low co
protein solubility is decreased by high salt concentration ed to
sulphate is one of the most soluble salts. When it is ac
protein, some proteins precipitate at a given 58
Chromatography. “4 eral types of this procedure1
SUSE
= partition chromatography, !
- “{on-exchange chromatography; |
- adsorption chromatography’; Ae
- ‘affinity chromatography. oa s¢
Electrophoresis. When current is Lpplied tomolecules in ters ae a
with a net negative charge (anions) migrate fib the anode an nye
positive charge towards the cathode. There are several types of electrophoresis ).
zone electrophoresis, discontinuous electrophoresis (for short disc-electrophoresis.
isoelectric focusing.
: dn vo
[hy
city of inter action |of. antibodies. with.
—
the corresponding antiserum
met from phim be animalHr to immunizationW
with thiss protein.
Ultracentrifugation (see molecular mass of proteins).
SIMPLE PROTEINS
Simple proteins contain only amino acids. There are several groups of
simple proteins.
~~ Albumins. They are soluble in water where as most of proteins can be
dissolved in water only if low concentration of salt is added to the solution.
Albumins are
Shi onlyy by the liver. Es Ha protein:
ho of riser §
in the blood. This protein is r
hydrophobic molecules, such as fatty cid, eh wanspors of
drugs (e.g. salicylates. barbiturates, sulfonamides, penicillin, warf; of metals, ang
One of the major Finetions of albumin is its role in th ann, etc.)
oncotic pressure of plasma...- € maintenance of the
: Globulins. They are insoluble iin water, but solublei d
Globulins include several fractions: ay, ca-, 3-, an Ppelobalis dilute salt solutions.
oq-Fraction contains several proteins, e. ¢. HDL (high
2nd ay-antirypsin. ensity lipoproteins)
a-Fraction contains haptoglol
Zznsity lipoproteins. §
B-Fraction contains transferring
linoproteins). 3 prothrombin, ang LDL (low
~-Globulin fraction contains immunoglobulins,
There are five classes of immunoglobulins: M,
participate in defence reactions against antigens, » Gy A, E, and D. They
AN Eb
obin, ceruloplasmin, and VLDLp (very 1
Cry low
Ki
density
! [i
V2 Shs Yad

fo J
- luble in
3 % : erm. They are = are
Protamines. These proteins were found mn oe lysine residues; and fsios
Protamines contain large number of argiuyne pd dic pro nt of
strongly basic proteins. Therefore they tion of insulin used for treate
Protamine zinc insulin is a commercial preparatio
diabetes mellitus.
Histones. Like protamines, they ively ©
number of arginine and lysine residues and _- PIA fo play
located in the nucleus of a cell and are linked Wi
and folding of DNA within the nucleus. ; .80 % alcohol, but
Prolamines and glutelins. They are soluble n 7 eed corn, gliadin
insoluble in water. These proteins are of vegetal origin, il Z
and glutenin from wheat, hordein of barley, oryzenin ot rice. utions
Scleroproteins (or keratinoids). They are insoluble in water, sat oo
and organic solvents, but are soluble in hot strong acids. These pr 4 tender:
structural function, e.g. collagen in connective tissue, bone, cartilage, an py
keratin of hair, skin, nails, horn, and hoof; elastin in the artery wall and ligaments.
water.
ey also contain large
: e
harged. Histones ar
a role in packing
are basic proteins; th
-
TT hetk eS CONJUGATED PROTEINS
Conjugated protein consists of a simple protein combined with a non-protein
component. This non-protein component is called prosthetic group. Conjugated
proteins are classified according to the nature of their prosthetic groups (Table ).
4 Chromoproteins. These are proteins with colouired prosthetic groups.
Representatives of this group of conjugated proteins are divided into a)
hemoproteins, b) flavoproteins, and ¢) magnesium porphyrins.
*. Hemoproteins contain heme of red colour. Representatives of
carry out different functions in the body, e.g. hemoglobin
of O; and CO; in the blood, myoglobin transports O, in
participate in energy production.
: proteins contain riboflavin (vitamin B,) of yellow colour
included into flavoprotein as derivatives (co-¢ es : . . j
(FAD) and flavin mononucleotide (FMN). These dophyil nine dinucleotide
and FMN) are components ‘of oxidoreductases, ie e pw B, (FAD
oxidation-reduction reactions in the cells, e.g. FAD (FMN)-deponnt at catalyze
Magnesium porphyrins. The representative of this groy ydrogenases,
responsible for the solar energy fixation in green plants 1s chlorophyll, 1t is
hotosynthesis by catalyzing the Slaavape Wh
: Nucleoproteins. These 6 Clas '3ge
of water into hydrogen and ox
: hemoproteins
Is Involved in transport
the cell, cytochromes
; the vitamin is
combined with positively charged histone Th
: ; - here gre
deoxyribonucleoproteins (DNP) and ribonucleoppor. proteins:
DNA as prosthetic group and are | th Ins (RNP). pnp contain
mitochondria. RNP tontain RNA, © nucleus (chromosomes) and
in cytoplasm (ribosomes). DNP ang € nucle
toplasm are as ! and nucleoli, g¢
containing viruses, respectively. O present in p

-
: Table © Conjugated proteins.
Cl
Name of conjugated protein | Prosthetic group Examples |
Chromoproteins:
a) Hemoproteins Fe, heme Hemoglobin, myoglobin,
cytochromes i
b) Flavoproteins FAD, FMN FAD (FMN)-dehydrogenases |
c) Magnesium porphyrins Porphyrins Chlorophyll “
Nucleoproteins - DNA, RNA Chromatine, ribosomes
:
i
Lipoproteins’ Lipids Chylomicrons, VLDL, LDL, |
HDL |
Phosphoproteins H;PO, Caseine (in milk), regulatory i
phosphoproteins in cells
| Glycoproteins Carbohydrates Proteoglycans, glycoproteins !
of the blood, mucin
Metalloproteins Cu Hepatocuprein, ceruloplasmin |
| Fe Ferritin, hemosiderin,
transferrin, many enzymes
> ¥ Lipoproteins. These are proteins combined with lipid components
(cholesterol, phospholipids, triacylglycerols). Lipjproteins are present in blood
plasma and in cell membranes. Lipoproteins participate in the structural
organization of myelin sheath. Lipoproteins of the blood plasma are divided into
chylomicrons, VLDL, LDL, and HDL.
+ Phosphoproteins. They contain H;PO, residue. The phosphoric acid is
“added to the hydroxyl groups of serine and threonine of proteins. Phosphoproteins
/ are present as nutritious proteins in milk (casein), egg yolk (vitellin), and fish
spawn (ichtulin). Phosphoproteins are abundant in the CNS.A number of enzymes
that take part in regulation of metabolism can exist in both phosphorylated and
dephosphorylated forms. Phosphoproteins are formed due to phosphorylation of a
protein by protein kinase with participation of ATP. Dephosphorylation of the
protein is catalyzed by protein phosphatase.
det LEER A IE A Sd EE a TEER gl]

L>
£4
Be Glycoproteins. The prosthetic groups of glyco
carbohydrates and their derivatives: glucose, galactose, mannose, hexosamines
(glucosamine and galactosamine), glucuronic acid, neuraminic acid, sialic acid.
The prosthetic group of certain glycoproteins may include
glycosaminoglycans (also called mucopolysaccharides), e.g. such as hyaluronic
acid and chondroitin sulphuric acid. These glycoproteins are named proteoglycans
and are present in extracellular substance of connective tissue.
Blood group antigens and many blood plasma proteins (except for
albumins) are glycoproteins, e.g. interferon, some hormones (gonadotropic and
follicle stimulating hormones). Saliva and secretory products of mucous
membranes (mucus) contain glycoprotein mucin.
Metalloproteins. They contain metal ions, E.g. ferritin, transferring,
hemosiderin contain non-heme iron. Ferritin serves for storage of iron (iron
depot); transferrin for transport of iron; hemosiderin is water-insoluble iron-
containing protein which is deposited mainly in the liver and spleen and slowly
releases iron when deficiency exists.
Ceruloplasmin is the major transport protein for copper; the protein
transports copper from the liver to peripheral tissues. Hepatocuprein is present in
the liver.
Many enzymes are also metalloproteins,
proteins are represented by
BIOLOGICALLY IMPORTANT PEPTIDES
On definition, polypeptide chains containing less than 50 amino acids are
called peptides (polypeptide chains containing more than 50 amino acids are called
proteins). Peptides have significant biological activity and exhibit a number of
specific functions.
Natural biologically important peptides occurring in the organism are
divided, depending on their on their origin and functional specificity,
groups.
- 1) Regulatory peptides:
vasopressin (stimulates the kidney to retain water);
oxytocin (stimulates contraction of uterus during childbirth and the ejection
of milk by mammary glands during lactation;
corticotropin (stimulates synthesis and secretion of glucocorticoids by
adrenal cortex);
glucagon (stimulates glucose production in the body);
calcitonin (decreases calcium and phosphate level in the blood);
melanotropin (stimulates synthesis of pigment melanin);
hypothalamic releasing factors (stimulate synthesis and secretion of
hormones in adenohypophysis).
2) Peptides of GIT, or prptides involved in digestion:
gastrin (stimulates the secretion of HC in the stomach);
secretin (stimulates secretion of alkaline pancreatic juice, rich in
bicarbonates);
into several
1
HH
BE
ETE
EEE
NEED
NEN
EE
NEW
NES
-
ne . o) i
MAAC Nav [ETA [EE aed
»

<
) tic juice rich
sholecystokinin, or pancreozymin (stimulates BF eli): J
in enzymes but depleted in bicarbonates; also increases the bi i
“ation. inhibits smooth
vasoactive intestinal peptide, VIP (causes vasodilation, inhi
muscle contraction).
—7 3) Vasoactive peptides:
. sure);
angiotensin (is a powerful vasopressor, thus increases blood pres )
vasoactive intestinal peptide (causes vasodilation);
bradykinin (causes vasodilation);
atrial natriuretic factor (decreases blood pressure, causes vasodilation,
Increases sodium excretion in the urine);
kallidin (causes vasodilation).
4) Neuropeptides. They are produced in the CNS.
Representatives include opioid peptides such as endorphin and enkephalins
(relieve pain, produce pleasant sensation and ca
(takes part in mechanisms of sleep); substance P (stimulates perception of pain, an
action that is opposite to that of opioid peptide 5); number of neuropeptides are
involved in the biochemical mechanisms of mem ory, fear, learning etc.
Some peptides do not belong
to any type of bi
c= : gas] and i
glutathione plays a major role in the defince of AS Betoxides. Also
‘oxidative damage and thus protects prot ei «ry
activity, Is from the logs of their biological
use euphoria); ¢ (sigma)-peptide

PROPERTIES OF ENZYMES
. Almost all biological functions are based on chemical reactions catalyzed by
enzymes. Enzymes are proteins that catalyze specific reactions, i.e. enzymes are
catalysts of biochemical reactions.
Enzymes exhibit a number
of properties. _
1. Enzymes accelerate the rate of chemical reactions but do not change the
equilibrium,
2. An enzyme (E) cen interact with a tiolecule called substrate (S). An
unstable iritermediate enzyme-substrate complex (ES) is formed and then it
decomposes 16 release the enzyme and'to yield the product of reaction (P):
E+ S =ES—>P + E.
After the termination of an enzymatic reaction, the enzymes remain in an
unaltered state, i.e. after the release of enzyme from ES complex, enzymes are
* capable of reacting with new molecules of substrate.
3. The substrate molecules involved in enzymatic reactions are of smaller
size in comparison to the enzyme molecule.
4. The enzymes exert their action when preseiit evén in negligibly small
concentration. Even a single molecule of enzyme can convert all:the substrate
molecules into product, oC
5. Enzymes often work in series, in pathways
or in cycles, so that the
product of one enzyme is used as substrate by the riext enzyme to produce a new
product. In turn, this new compound can be used by the third enzyme to yield the
third product:
E
A—>B Zc p By p By
6. Enzymes accelerate the rate of chemical reactions under physiological
conditions: moderate temperature (body temperature, 37°C), pH range close to
neutral; at an atmospheric pressure, and in aqueous environment.
7. The process of enzymatic catalysis is carried out not with the whole
molecule, but only with its active centre. |
8. Some enzymes may contain
so called allosteric (regulatory) centre.
9. Enzyme exhibit specificity of action. :
10. The velocity of an.enzyme reaction depends on the concentration of a
Substrate, the concentration of an enzyme, temperature, pH, and ‘the concentration
of the reaction product. (These are so-called factors affecting the enzymatic
reaction rate: t°, pH, [S], [EJ], [P]).
11. Many enzymes require cofactors to catalyzea reaction.
12. Enzymes can accelerate the reaction rate by decreasing the activation
energy of the reaction.

~. LE 2
; i me depends OT
13. The rate of enzymatic reaction; "e: the activity i irs oecelerate
the presence of activators and. inhibitors in the medium.
the reaction, the inhibitors.retard the reaction.
aN i
™ 3 i TT av S
TBRIC CENTRES’ OF ENZYMES
LE Ce. sdination of amino
The active centre of an enzyme represents 2 ume aR eraction of
acid residues in the enzyme molecule that provides for ot «t. The active centre
enzyme with
the substrateto convert the substrate
into POPE © sg pormed
takes up ‘only :a small-portion of the whole enzyme. ug nal conformation
during folding of the -enzyme into its: patticillar three-dicensio the nature of the
(tertiary structure), The structure of the active centre depends on the ically and
substrate and corresponds to the substrate structure both geome ! - » ny
electrostatically. Usually, the active site is sitnated in the crevice of CI 0
enzyme molecule. : : per.
i The active centreis subdivided
into two partsy binding site and ¢catalytic ste.
The binding ‘site. Substrate binds to this site-on the enzyme the chemical
changes will‘take place. E PR i»
The catalytic site is directly involved in’ chemical interactions with the
substrate arid conversion of substrate info product.
“47 ACTIVE AND ALLOS
FED SEA a we
ia”
The allosteric centre
is designed for the binding: of certain, low-molecular
compound whichis named effector or modificator. The binding of an effector to
the allosteric centre causesa change in the tertiary and quaternary structure of
protein molecule.
This; in turn, leads
to a change. conformation of the active centre
and results in aa increased or decreased enzyttie activity. Thus, allosteric centre
can regulate the enzyme activity,
The enzymes which activity is controlled by the state of Both the active and
the allosteric centre are called allosteric enzymes. aE
SPECIFICITY OF ENZYMES +7
Enzyme. specificity is one of the most important properties of
aT Ll PRN 0s oren :
high specificity of enzymes Is determined by the conformation] ie
electrostatic complementarity between the molecules of enzym and subst an
and. 15 also based on the re Chih active centre of hy strate,
: Specificity of ‘action provide] for the selective course os artical é enzyme.
reaction” among the- vast variety of different cheinical rea LL chemical :
simultaneously occur
in the living cells: To AL reactions; which -
Most enzymes are highly specific'for
both the type of § a
and the pature of the substrate.
So, Pin ed omni catalyzed
specificity. € Specilicity
and reaction

Substrate specificity
There are several types of substrate specificity.
a) Absolute specificity.
Many enzymes exhibit absolute specificity, i.e. these enzymes can recognize
only a single compound as a substrate and ‘are able of catalyzing the conversion of
a single molecule only. E.g. arginase splits arginine; or urease catalyzes
degradation of urea. :
b) Relative or group specificity.
Some enzymes can recognize a group of molecules with similar groups of
atoms or the same types of chemical
bonds in the substrate molecule.
This is 50
called relative (or group) specificity. E.g. pepsin (enzyme involved in digestion)
cannot split carbohydrates or lipids, but can split any protein of animal or vegetal
origin.
c) Stereochemical specificity.
This type of specificity is associated with the existence of stereoisomers.
Stereoisomers are the molecules that are almost identical; they contain the same
number and types of functional groups but differ in the orientation of these
chemical groups, e.g. L- and D-forms of amino ‘acids or cis- and trans-forms of
chemical compounds. Oxidases of L-amino acids can convert only L-amino acids
and do not act on D-amino acids, Fumarase catalyzes conversion of fumaric acid
only (¢rans-isomer) and do not convert maleic acid (cis-isomer).
Reaction specificity
Some enzymes can recognize the type of the reaction catalyzed. E.g. amino
acid histidine can undergo deamination (the release of the amino group) or
decarboxylation (the release of carboxyl group) by two different enzymes, which
are specific only to certain type of'the reaction, i.e. histidine ammonium lyase or
histidine decarboxylase, réspectively.
SIMPLE AND CONJUGATED ENZYMES) 25)
COFACTORS OF ENZYMES =
There are two types of enzymes in nature: simple and conjugated enzymes.
The simple enzymes compose of amino acids only. The examples of simple
enzymes may be hydrolytic enzymes, such as pepsin, trypsin, ribonuclease,
phosphatase; and some others.
;
Most natural enzymes are conjugated enzymes: they compose of both
polypeptide chain and a non-protein component. The polypeptide chain of them is
called apoenzyme. The non-protein
ERM 1s called a cofactor. The whole
molecule of a conjugated enzyme containing both apoenzyme and cofactor is
called the holoenzyme.
Holoenzyme = Apoenzyme + Cofactor
Poly pent de.

: 4.
Cofactors may differ in the St IB df
chain, If the cofactor and apoenzyme ion ha
the cofactor does not dissociate" casi % dh
called the prosthetic group.. If the chemi eootor dissocia
apoenzymeé
are weal, non-covalent, and the co |
apoenzyme,
such a cofactor is called a coenzyme.
Cofactor
Prosthetic Coenzyme
group
Apoprotein- without its cofactor exhibits no enzymatic aoltviep J :
because both prosthetic group and “coenzyme actively participate in C emica
reactions. a i ) | ary i Cs
‘Many metal ions can be cofactors of various enzymes, e.g. such ions as Mg |
(glucokinase), Mn®* (arginase), Ca” (amylase), Fe** (cytochromes), Zn
(carboatihydrase), Cu’ (cytochromoxidase). -
~ Coenzymatic functions of vitamins
3 me LR
are so
Table . Active forms of vitamins.and their functions. .
Vitamin Active (or coenzymatic) form. Biological function
B, (thiamine) TDP (thiamine diphosphate) ~~ | Deshi” ol
A TTR ’ Lit i! or-ketoracids
B, (riboflavin) [FAD (flavin adefiine dinucleotide). | Hudvmoos vr.
| FMN{(flavin'mononuclectide) ®) Hydrogen transport
By Gyridoxing) |PLP Gytidosal Phosphate), PMP |Transamiag
© |Cvidotmine phosphate) | gocarpenon 5
amino-acids
PP (nicotinic acid, [NAD (nicotinamide pars :
nicotinamide) dinucleotide), NADP (iicofinn mine Hydrogen transport

4
adenine dinucleotide phosphate)
B; (pantothenic |HSCoA (coenzyme A) Transport of acyl
acid) groups,
Folic acid (By) THF (tetrahydrofolic acid) Transport of one-
carbon units
~ MECHANISM OF ENZYME CATALYSIS ‘1
In enzymatic catalysis, an enzyme (E) binds reversibly with its substrate (S) to
form an unstable intermediate enzyme-substrate complex (ES) which, on
completion of the reaction, decomposes to release the enzyme and to yield the
product of reaction (P):
E+S =ES—>P + E.
The interaction between enzyme and substrate is of very short duration but
the rate, or velocity of the enzymatic reaction is very high. The mechanism of
enzyme catalysis helps understand how enzyme works. -
Substrate binds with the active centre of enzyme and is oriented in such a
manner that increases the probability of product formation.
- There aré two models that can explain the enzyme action.
1. The lock-and-key model (Fisher’s model) assumes that the three-
dimensional structure of the active centre of the enzyme is complementary to the
structure of the substrate, i.e. substrate fits to the enzyme similar to lock and key.
This complementarity (or exact matching), both spatial and electrostatic, allows
the substrate to enter and interact with the active centre of the enzyme.
2. The induced-fit model (Koshland’s model). According to it, substrate
does not fit exactly to the active centre of the enzyme. The binding of the substrate
to the enzyme induces a change in the three-dimensional structure of the active
centre and causes the unique conformational orientation of specific functional
groups at the active centre. As a result of these interactions, the shape
of the active
centre fits the shape of the substrate.
The basic principles of an enzyme-catalyzed reaction are the same as any
chemical reaction. At the initial state all molecules possess certain amount of
energy. Chemical reactions can proceed fast enough when the substrate (chemical
compound) gains the activation energy. The activation energy is an energy which
is needed to be supplied to one mole of a substrate to transfer all its molecules.to
the activated state. I.e. activation energy is the energy required to convert all
molecules of a substrate from the initial state to the transition state and to trigger
(launch) a chemical reaction.
At the transition state, molecules of substrate have an elevated free energy;
they become more active and capable for conversion to some other molecule. In

the reaction to proceed is ed supplied n
i :s is not suitable becd
ving organisms, this 15 nO se
pr py elle = delicate ‘biological ov do A ative
denaturation. Enzymes help to solve the problem: tney P
‘raaction to occur.
reaction pathway that requires less energy for the reacts
. :ng the activation
Enzymes accelerate the chemical FRActio 1¥ omy re ‘fuce the energy
energy of the reaction.
Due to binding with substrates, €0 rated ecules which
of transition state by making increased the mymber of segivaied 105 or without
become reactive at lower energy level (i.e. at body temp
‘additional heat. )
the laboratory, the energy required for
Free
energy
Final state
>
Progress of reaction
Fig. Lowering of activation energy by enzyme. § substrate; P = product;
A — the transition state of a non-enzymatic reaction; B ;
the transition state of a
enzymatic reaction; C — energy level of substrate; D — energy leve] of product; ABs
(or C to B) — the activation energy of an enzymatic reaction; ABne (orCto A) _ the
activation enetgy of a non-enzymatc reaction; B to 4 lowering of activation
energy by enzyme, The conversion of substrate to product demands that all the
molecules of substrate reach a transition state. In'the transition state molecules are
most reactive. Enzyme-catalyzed reactions proceed more readi °
= ¢ . : ly because the free
energy of the transition state is considerably lower for the enzyme ogi? e |

wv
IS
ry
rh
/
(NE
CLASSIFICATION AND NOMENCLATURE OF ENZYMES.”
It is estimated that there are about 10,000 enzymes in the human body.
Enzyme classification is basd on the type of the reaction catalyzed. According to
the accepted classification, the enzymes are divided into 6 classes (see Table ).
Table . Classification of enzymes.
| Class of | Type of the reaction | Scheme of the| Examples of
enzymes that they catalyze | reaction enzymes
1. Oxido- Oxidation-reduction | AreqtBox Agx+Breg | Dehydrogenase,
reductases reactions; transfer reductase,
of protons and/or cytochrome,
electrons peroxidase,
catalase
2. Transferases | Intermolecular A-B+C— Acyltransferase,
transfer of radicals | A +B-C glycosyltransferase,
kinase,
methyltransferase,
aminotransferase,
; phosphotransferase
3. Hydrolases | Hydrolytic A-B +H,0— Esterase,
reactions: cleavage | A-H + B-OH glycosidase,
of molecules with
use of water to
peptidase (such as
pepsin, trypsin-and
break chemical chemotrypsin),
bonds phosphatase
4. Lyases Cleavage of various {A-B—> A+B Dehydratase,
groups from | decarboxylase,
substrates without “| hydratase,
use of water, deaminase,
cleavage’such bonds f synthase
as C-C, C-O, and
C-N; removal of
H,0, NH; or
CO,with formation
of double bonds or
addition of these
chemical groups to
double bonds
5. Isomerases Isomerization AZ IsoA cis-trans
reactions: isomerase,
| rearranging of mutase,
atoms within a epimerase,
molecule without racemase,

ee]
change of 1¥
composition
6. Ligases Energy-dépendent
(synthetases) synthetic teactions, | /
ie. bond formatiof
between two
molecules with use
lcleavage lL. ————
has formal specific
number (B.C. number, or Enzyme * r This E.C. a
represents a four-digit code, and each partis sepatat 2d by a dot from each © 3
(e.g: 2.1.1.3). The first digit denotes the class (one of the SIX classes men one
above) to which the enzyme belongs. The second number indicates sub-class
which characterizes the substrates involved in the given type of chemical
conversions. In turn, the sub-classes are
designate the nature of donor or acceptor compounds that participate in the
© reactions of the given sub-class. The sub-sub-class number takes the third place in
the enzyme code. Finally, each enzyme has the ordinal number within the sub-sub-
class (the fourth position in the code number).
Enzymes are usually named by adding the suffix “-ase” to the name of their
substrate or to a word or phrase describing their activity. But for many enzymes, 2 ~
trivial name is more commonly used, e.g. pepsin, trypsin, cytochromes,
etc.
ISOENZYMES “1 +
Isoenzymes (or isozymes) are the group of enzy: _
reaction-but differ in their amino acids sequence nd psalm the same
properties. Isoenzymes differ in:
: many of their
- molecular mass;
- their primary, secondary, tertiary and (if it ex
quaternary structures; ry and (if it exists for this enzyme)
. affinity for substrate;
- enzyme activity;
- electrophoretic mobility;
- pH optimum;
- regulatory properties;
- charge;
_ localization if different tissues;
- response to activators and inhibitors;
- stability against the action of denaturation a .
variety of subunits composing the overall ¢ Bee
For example, lactate dehydrogenase
co rie ac : DH) ;
pyruvic acid into lactic acid. This enzyme rn the conversion of
- - Tour subunits of two
of energy from ATP |
J
divided into sub-sub-classes which

vardl
4
different types, H (heart type) and-M (muscle type). Five isoforms of this enzyme
(five isoenzymes) may exist due to the following combinations of subunits:
HHHH, HHHM, HHMM, HMMM, and MMMM, which correspond to the
isoenzymes LDH,, LDH,; LDH; LDH; ‘and LDHs. The isoform LDHis
predominant in the heart muscle and the LDH; form in skeletal muscle and in the
liver.
serum. B.g. the activity of LDH,
as well as creatine kinase MB form
is increased
in
the blood serum in myocardial infarction, and LDH; in hepatitis.
(8 4 G
KINETICS. OF ENZYMATIC REACTION: @9)
Enzyme kinetics is the quantitative characteristics of enzyme catalysgs. The
enzymatic reaction may be described as the following chemical equation:
SS +E T2ES
—> P + FE,
Le. an enzyme (E) reacts with: Substrate?(S)
to form an intermediate enzyme-
substrate complex (ES) which further dissodiates into the free enzyme and the
product of reaction (P). HE wn AE
The relationship between
‘the’ enzfimiatic reaction rate and, the different
substrate concentrations
may be! described with the hyperbolic curve (Fig. ):
V A
Vmax CR Tr
Viasi
2
Fig. Michaelis — Menten plot of velocity (V) versus:substrate concentration
[3]. Vinax — maximal velocity of the reaction; Ky, — Michaelis constant, "
As it is seen from the plot, at low substrate. concentrations, the rate of the
enzymatic reaction is directly proportional to the substrate concentration .[S], At
high substrate concentrations, the reaction rate reaches a maximum and becomes

AEE
-
PN
he) P-
i veloci
H
alg i THe maximum
constant independent of the substrate concentration 15] rated with substrat®. 7
attained when all the active yi y pp pe i gnated a Eh ond
ay ‘by 2, the. oinit Vmax 14 2%" 7g vperbo
draw lr ih aint o tie crossing with the IWRC (Kr) M2Y be.
then direct it downright, a point thatis called Mictize fs oo ds the substrate
"designated
at the horizontal axis. Thus, Michaelis cons nzymatic reaction is half
. H ee
concentration (mol/litre) at-which the velocity arih
the imu value. iit AEEVery
he Kis ory important for the characterization OF CPE os foveer
is an indication of the efficiency of substrate binging $ other words, the Kum.
the K,, the higher efficiency-of enzyme-subsirate binding the K... the greater the
reflects the. affinity of enzymes for substrates: the lower - N cy i ;
affinity
of the enzyme for the substrate
(for ES complex formation).
Michaelis and Menten also derived the:equation
enzyme. The Km
= Via [81 © where: -
Kn¥ (S]
¥V ac — maximum velocity that the reaction can attain.
This equation, named the Michaelis-Menten equation, is very useful in
characterization of enzyme behavior E.g: when [S] is equal to Kn; the
denominator in the equation
is equal to 2[S],
and the V becomes equal t0- Vmax:/2.
As K,, is specific characteristics of any enzyme, it is very important to know
Kn of every enzyme. To determine Ki, it ismiecessary to calculate Vay, and divide
this value by two, However, it is very difficult to determing quite accurately the:
: Vmax value from a hyperbolic-eurvs erefore; Lineweaver
and Burk suggested the:
[ ~ . double-reciprocal plot to transfo olic curve plot into a straight line:
(linear) plot (Fig. ). ]
1
— A
v | 1)
co ie 1 _ [<r / 1 SN AT
Vv EN
—
SA
Uma
hynes woo fF plot
| J Vmax : § dl } 5 km
; ) -> == 5) 4 27
yy Vv a) ; Umax
a | (8)
Fig. . Lineweaver
Burk plot. 1/V - the resiprosal of.
1/[S] — the reciprocal of the substrate concentration roca of the reaction velocity;

-H-
TR
&
In the Lineweaver-Burk plot, the intercept on the vertical axis equals 1/ Vp,
The intercept on the horizontal axis equals—1/X,, ~~".
DEFINITION OF ENZYME ACTIVITY (QUANTITATIVE ESTIMATION
"OF ENZYME CONCENTRATION). UNITS OF ENZYME ACTIVITY
Quantitative estimation of enzyme concentration is based on measuring the
rate of the reaction catalyzed by the enzyme. This is done under standard
conditions. At optimal temperature, pH, and full saturation of enzyme with
substrate, the rate of enzyme-catalyzed reaction is directly proportional to the
enzyme concentration.
The enzymatic reaction rate may be measured by two ways:
1) by the rate of substrate disappearance in the solution (substrate depletion
rate), or
2) by the rate at which the reaction product is formed.
The rate of enzyme reaction is defined as an énzyme activity.
There are several units to express the concentration of an enzyme and
quantitatively estimate enzyme activity.
International units (IU). One IU is defined as the amount ofEo
convert 1 pmole (micromole) of substrate per minute under standard conditions
(at optimal temperature and pH), i.e. pmol/min,
Katal (kat). One kat is the amount of snzyme that converts 1 mole of
substrate per second, i.e. mol/sec. :
lkat=6" 10” XU, or
1 IU= 16.67 nkat.
Specific activity is the ‘number of international units per milligram of
protein (IU/mg protein), or kat/kg protein.
Molar aciivity (the turnover number) is the number of moles of substrate
converted to product each second per mole of enzyme (for easier
memorizing, S$ —>P/"/E).
FACTORS. AFFECTING THE ENZYMATIC REACTION RATE:
TEMPERATURE, pH, SUBSTRATE AND ENZYME RY
CONCENTRATION ’ ei
Enzymes are dynamic molecules, and the ability of enzymes to convert
substrates into product may be changed under different conditions.
1) The effeet of temperature on enzyme activity (thermolability of
enzymes). o
Enzymes are sensitive to temperature changes. A rise in temperature —
exceeding 40-50° C makes the reaction rate increase, As temperature is increased,
Tore molecules get activation energy. The increase in the reaction velocity is due
to an increase in the number of molecules that have suff; cien enter into
the transition state. t energy to

. ivity due. 10")
{ At a temperature above 50° C the enzyme losed its 8SviEY $
denaturation, and enzymatic process stops (Fig. ): 5
Optimal temperature }
-
[1]
[1]
|]
[J

’
)
]
|
1
'
'
’
1
[] —
>
0 37-45 100 tC
Fig. Effect of temperature on enzyme activity (on the rate of an enzyme-
catalyzed reaction).
Temperature 40°C (that is close to body temperature) is optimal: at this
perth, enzymes operateat maximal efficiency. At low temperatures (0° C)
Zymes undergo no denaturation or destruction, although their activity goes down
nearly to zero.
4 The effect of pH on enzyme activity.
nzyme activity depends on pH (hydrogen ion i nzym
has an optimal pH value at which their activity is maximal at higher oe Loe H,
activity decreases. Usually enzymes have their high activity within a na an
of PH, which corresponds to the physiological medium PH values of 6.0.8 An
exception
is pepsin, for which the optimal pH is 1.52.0 si % pe on
optimalffies within an alkaline interval (7.5-8.5 . nid Sr a
phosphatase (optimum pH 4-5), and alkaline phosphatase po en 8, mcid
The graph of the reaction rate versus pH is re pumum pH 9-10).
curve (Fig. ). presented by a bell-shaped
Va Optimal pH
Fig. . Effect of pH on the e CI
ZYME activity (ve]oo; ity of the reactjo n).

—
ww
“
*
~-13 a
Hydrogen ion concentration (the pH) can influence catalytic activity of
enzymes due to capability
of hydrogen ions to change ionization of the amino acids
at the active centre of enzyme and thus change or stabilize the active conformation
of the enzyme.
3) The effect of substrate concentration on enzyme activity.
If the enzyme concentration
is constant, the velocity of enzymatic reaction
increases with the increase of the: substrate concentration until the maximal
velocity, Via, is attained. The maximal velocity is attained when all the active
sites of the enzyme are saturated with substrate.
At low concentrations of a substrate,
as the substrate: concentration increases,
the velocity will also increase. But as the substrate concentration gets higher and
higher, this curve becomes hyperbolic and enzyme approaches the maximum
velocity. The further increase in substrate concentration produces no effect on the
reaction rate (Fig. ).
V4 |
Vine fevwoenemnanconcnne
[E]is constant
[S]
Fig. .The effect of substraté-concentration on reaction rate.
4). The effect of enzyme concentration on enzyme activity,
The reaction rate is proportional tothe concentration of the enzyme, if the
substrate concentration
is constant. There is a linear relationship between enzyme
concentration and enzyme velocity (Fig. ).
v
V4
[8] is constant
E]
Fig. .The-effect of enzyme concentration on reaction rate.

ENZYMES
an enzyme,depends ol
celerate the reaction,
FAG) | . ON OF
© 52 4cTIVATION AND INVHBIT ;
ha s jvity ©
. ction, i.e. the actiVl
The rate of a5 euzymatl® BF hibitors. Activators aC
the presénce of activators
inhibitors retard reactiofl.
Activators
etal ions as sho
The most common activators are represented by m
Table
Table . Metals as activators of enzymes
Metal ion Metal activated enzyme
Fe™ Cytochrome
Catalase:
Peroxidase
Zn Lactate dehydrogenase
Carbonic anhydrase
[ Mg? Kinases
Mn?* - Arginase
Mo™ | Xanthine oxidase
Cu’ “Cytochrome
Ca Amylase
Lipase
The role of metals in the activation of enzymes
Metal ions may
They may:
1) be a prosthetic group for, certain enzyme
2) act as donors and acceptors of electrons;
3) facilitate the attachment of a sub
4) combine with the substrate to fo
5) participate directly in the forma
6) stabilize the three-dimengional
7) play the role of allosteric modu
perform various functions within the enzyme molecule.
ons;
Strate to the active centre;
I a true Substrate;
’
tion of the active centre;
Structure of the enzyme;
lators or effectors,
Anions exert minor activation &
I 8Clvation gffect
" ings on + NzZym 3:
salivary amylase; chloride ions are their active, ©: Exceptions re: pepsin and
rs.

RTE
Inhibitors. Types of inhibition
There is irreversible and reversible inhibition.
Irreversible inhibitiofi. Irreversible inhibitors bind tightly, with a stable
covalent bond, to the functional groups of an enzyme, and this often leads to
complete inactivation of enzyme. Many toxins and- poisons, are irreversible
inhibitors and act on the organism according to this mechanism.
For example, ‘the death due to poisoning with hydrocyanic acid comes
because of complete inhibition of the respiratory enzyme cytochrome oxidase of
the cerebral cells. Todoacetate inhibits enzymes having HS-group in their active
centre. Di-isopropyl fluorophosphate (DFP) inhibits enzymes with hydroxyl
+ ---groups.in-the active.centré..—.........
Reversible inhibition is divided into competitive and non-competitive
inhibition.
In the ‘competitive inhibition, the: inhibitor is a structural analogue of the
substrate, i.¢. inhibitor has a structure similar'to the structure of substrate, and this
inhibitor competes with the: substrate for the binding to the active centre of the
enzyme. When the inhibitor binds with the active centre of the enzyme it prevents
binding of the substrate to the enzyme, and the enzyme-inhibitor complex is’
formed. But, unlike the enzyme-substrate complex; this enzyme-inhibitor complex
does not undergo degradation, and the product'of the reaction
is not produced:
E+ 8S =2E—>P + E :
I + § ==21S —> No product.
An increase in substrate concentration may reverse the inhibition. If
substrate concentration is enormously high, when compared to that of inhibitor, the .
inhibitor is replaced by the substrate molecule in the EI complex (enzyme-inhibitor
complex), and inhibition is reversed. Pharmacological action of many drugs
(medicines) may be explained by the principle:of competitive inhibition.
The effect of a competitive inhibitor on the activity of the enzyme. may be
demonstrated by Michaelis-Menten and Lineweaver-Burk plots (Fig. ).
Vet : Vinx =Vi
2 )
7
/"
Kn K; [S]
Fig. Competitive inhibition. Michaelis — Menten plot. 1 —in the absence of
inhibitor; 2 — in the presence of inhibitor.
Bi
SES
ET
EBT

fl Xi 3
LC
=
1 _1
i Vinx Vi —>
J 1 1
K, Ki (S]
the inhibitor: 2 — in the presence of the inhibitor. | )
The Be of a Rompetitive inhibitor on the enzyme reveals that Kn 1s
increased biit the maximum rate (Vmax) is not changed (remains the same).
In ‘the non-competitive inhibition the inhibitor has no structural
resemblance
to the substrate, and the inhibitor binds not to-the active centre but to
the other sites at the enzyme molecule (e.g. to the ES-comiplex). In many cases,
non-competitive inhibition turned out to be irreversible, and an increase in -
substrate concentration ‘does not reverse this inhibition. The effect of a non-
Ai
Co , wolot. 1 —i bsence of |
Fig. . Competitive inhibition. Lineweaver-Burk plot. 1 —1n the a C
competitive inhibitor on the activity of the enzyme demonstrated by Michaelis-
Menten and Lineweaver-Burk
plots is shown
in Fig: . o
V4
ER, 1 Vina
Bets gy AISRLI WPL,
|
Vi -
0 pomme frome op emeeecasccmiaam.
N. fi7
ilo
214 ,
a1
Fig. Non-competitive inhibition. Michasljs —
absence
of inhibitor; 2 — in the présence of inhibitor. Menten plot. | — in the
The effect of a non-competitive irhibitor
A L1 T On . .
the sane but Vig is decreased. the enzyme reveals that Kp, is

A
|
[S
ad
APPLICATION OF INHIBITORS IN MEDICAL PRACTICE
* Some inhibitors are used in medical practice as drugs for treatment of certain
Table
—_——
diseases. A few important examples are given in the Table
. Drugs used in medical practice as inhibitors of enzymes 3 >
om m—
~~
Drugs Mechanism of action Clinical
application
(the use for
treatment)
Trasylol --
Sulphanylamides
(sulphonamides).
5-Fluorouracil
L_
Acts as inhibitor for pancreatic enzymes
(trypsin, chymotrypsin), and kallikrein
Structurally, they are similar to para-
aminobenzoic acid (PABA). PABA is used by
bacterial cells to synthesize folic acid. Folic
acid is used by bacteria to transport one-carbon
“units for the DNA synthesis. DNA synthesis is
a basis for the cell divisiof¥i¢production of
bacterial cells. Hence, sulphanylamides act ag
competitive inhibitors: they block the enzyme |
that catalyzed synthesis of folic acid thus
leading to inhibition-of bacterial cell division.
Inhibits the enzyme thymidflate synthetase.
This enzyme produces TMP for the DNA
synthesis. When the DNA synthesis stops, the
cells stop dividing.
Acute
pancreatitis
Infectious
diseases
caused by
bacteria
Cancer

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