Download PROTEINS Proteins are unbranched polymers of amino acids linked

PROTEINS
Proteins are unbranched polymers of amino acids linked head to tail, from carboxyl group to amino group,
through formation of covalent peptide bonds, a type of amide linkage (Figure 1).
Figure 1: Peptide bond formation.
CLASSIFICATION OF PROTEINS
Proteins are classified:
1.
2.
3.
On the basis of shape and size
On the basis of functional properties
On the basis of solubility and physical properties.
1.
ON THE BASIS OF SHAPE AND SIZE
(a) Fibrous proteins: When the axial ratio of length: width of a protein molecule is more than 10, it is
called a fibrous protein. Example is α-keratin from hair, collagen.
(b) Globular protein: When the axial ratio of length: width of a protein molecule is less than 10, it is
referred to as globular protein. Examples are myoglobin, haemoglobin, ribonuclease, etc.
2.
ON THE BASIS OF FUNCTIONAL PROPERTIES:
(a) Defence proteins: Immunoglobulins involved in defence mechanisms.
(b) Contractile proteins: Proteins of skeletal muscle involved in muscle contraction and
relaxation.
(c) Respiratory proteins: Involved in the function of respiration, like haemoglobin, myoglobin,
cytochromes.
(d) Structural proteins: Proteins of skin, cartilage, nail.
(e) Enzymes: Proteins acting as enzymes.
(f) Hormones: Proteins acting as hormones.
3.
ON THE BASIS OF SOLUBILITY AND PHYSICAL PROPERTIES:
According to this scheme proteins are classified on the basis of their solubility and physical properties and
are divided in three different classes.
(a) Simple proteins: These are proteins which on complete hydrolysis yield only amino acids.
(b) Conjugated proteins: These are proteins which in addition to amino acids contain a non-protein
group called prosthetic group in their structure.
(c) Derived proteins: These are the proteins formed from native protein by the action of heat, physical
forces or chemical factors.
A. SIMPLE PROTEINS
These are further subclassified based on their solubilities and heat coagulabilities. These properties depend
on the size and shape of the protein molecule. Major subclasses of simple proteins are as follows:
1. Protamines
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These are small molecules and are soluble in water, dilute acids and alkalis and dilute ammonia and
noncoagulable by heat. They do not contain cysteine, tryptophan and tyrosine but are rich in arginine. Their
isoelectric pH is around 7.4 and they exist as basic proteins in the body. They combine with nucleic acids to
form nucleoproteins. Examples: Salmine, sardinine and cyprinine of fish (sperms) and testes.
2. Histones
These are basic proteins, rich in arginine and histidine, with alkaline isoelectric pH. They are soluble in
water, dilute acids and salt solutions but insoluble in ammonia. They do not readily coagulate on heating.
They form conjugated proteins with nucleic acids (DNA) and porphyrins. They act as repressors of template
activity of DNA in the synthesis of RNA. The protein part of hemoglobin, globin is an a typical histone
having a predominance of histidine and lysine instead of arginine. Examples: Nucleohistones,
chromosomal nucleoproteins and globin of haemoglobin.
3. Albumins
These are proteins which are soluble in water and in dilute salt solutions. They are coagulable by heat and are
changed to products that are insoluble in water and solutions of salt. The albumins may be precipitated (salted
out) of solution by saturating the solution with ammonium sulphate. Albumins have low isoelectric pH of
pI 4.7 and therefore they are acidic proteins at the pH 7.4. They are generally deficient in glycine. Examples:
Plant albumins: Legumelin in legumes, leucosin in cereals. Animal source: Ovalbumin in egg, lactalbumin
in milk.
4. Globulins
Globulins are insoluble in water but soluble in dilute neutral salt solutions. They are also heat coagulable.
Vegetable globulins coagulate rather completely. They are precipitated (salted out) by half saturation with
ammonium sulphate or by full saturation with sodium chloride. Globulins bind with heme, e.g. hemopexin,
with metals, e.g. transferrin, ceruloplasmin and with carbohydrates, e.g. immunoglobulins. Examples: In
addition to above, ovoglobulin in eggs, lactoglobulin in milk, legumin from legumes.
5. Gliadins (Prolamines)
Alcohol soluble plant proteins, insoluble in water or salt solutions and absolute alcohol, but they dissolve in
50 to 80 per cent ethanol. They are very rich in proline, but poor in lysine. Examples: Gliadin of wheat and
hordein of barley.
6. Glutelins
These are plant proteins, insoluble in water or neutral salt solutions, but soluble in dilute acids or alkalies.
They are rich in glutamic acid. They are large molecules and can be coagulated by heat. Examples: Oryzenin
of rice and glutelin of wheat.
7. Scleroproteins or Albuminoids
These are fibrous proteins with great stability and very low solubility and form supporting structures of
animals. In this group are found keratins, collagens and elastins.
(a) Keratins: These are characteristic constituents of chidermal tissue such as horn, hair, nails, wool, hoofs
and feathers. All hard keratins on hydrolysis yield as part of their amino acids, histidine, lysine and arginine
in the ratio of 1:4:12. The soft or pseudokeratins such as those occurring in the outermost layers of the skin
do not have these amino acids in the same ratio. In neurokeratin the ratio is 1:2:2. Human hair has a higher
content of cysteine than that of other species it is called α-keratin. β-keratins are deficient in cysteine and,
rich in glycine and alanine. They are present in spider’s web, silk and reptilian scales.
(b) Collagen: A protein found in connective tissue and bone as long, thin, partially crystalline substance.
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Insoluble in all neutral (salt) solvents. Is converted into a tough, hard substance on treatment with tannic acid.
This is the basis of tanning process. Collagen can be easily converted to gelatin by boiling by splitting off
some amino acids.
Gelatin is highly soluble and easily digestible. It forms a gel on cooling and is provided as diet for invalids
and convalescents. It is not a complete protein as it lacks an amino acid tryptophan which is an essential
amino acid.
(c) Elastins: These are the proteins present in yellow elastic fibre of the connective tissue, ligaments and
tendons. They are rich in non-polar amino acids such as alanine, leucine, valine and proline. They do not
contain cysteine, methionine, 5-hydroxylysine and histidine. They are formed in large amount in the uterus
during pregnancy. Elastins are hydrolysed by pancreatic elastase enzyme.
B. CONJUGATED PROTEINS
Conjugated proteins are simple proteins combined with a non-protein group called prosthetic group. Protein
part is called apoprotein, and entire molecule is called holoprotein.
1. Nucleoproteins
The nucleoproteins are compounds made up of simple basic proteins such as protamine or histone with Nucleic
Acids as the prosthetic group. They are proteins of cell nuclei and apparently are the chief constituents of
chromatin. They are most abundant in tissues having large proportion of nuclear material such as yeast,
asparagus tips in plants, thymus, other glandular organs and sperm.
Deoxyribonucleoproteins: It contain DNA as prosthetic group, are found in nuclei, mitochondria and
chloroplasts.
Ribonucleoproteins: It occur in nucleoli and ribosome granules. They have RNA as prosthetic group.
Examples: Nucleohistone and nucleoprotamine.
2. Mucoproteins or Mucoids
Mucoproteins are the simple proteins combined with mucopolysaccharides (MPS) such as hyaluronic acid
and the chondroitin sulphate. They contain large quantities of N-acetylated hexosamine (>4%) and in
addition substances such as uronic acid, sialic acid and mucopolysaccharides are also present. Water soluble
mucoproteins have been obtained from serum, egg white (α-Ovomucoid) and human urine. These water
soluble mucoproteins are not easily denatured by heat or readily precipitated by picric acid or trichloroacetic
acid. They have hexosamine and hexose sugars as the prosthetic groups. Mucoproteins are present in large
amounts in umbilical cord. They are also present in all kinds of mucins and blood group substances. Several
gonadotropic hormones such as FSH, LH and HCG are mucoproteins. Insoluble mucoproteins are found in
egg white (β-ovomucoid), vitreous humour and submaxillary glands.
3. Glycoproteins
Glycoproteins are the proteins with carbohydrate moiety as the prosthetic group. Karl Meyer suggested that
these proteins carry a small amount of carbohydrates <4 per cent such as serum albumin and globulin.
Carbohydrate is bound much more firmly in the glycoproteins than the mucoprotein. Glycoproteins include
mucins, immunoglobulins, complements and many enzymes. They carry mannose, galactose, fucose, xylose,
arabinose in their oligosaccharide chains.
4. Chromoproteins
These are proteins that contain coloured substance as the prosthetic group.
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(a) Haemoproteins: All hemoproteins are chromoproteins which carry haem as the prosthetic group which
is a red coloured pigment found in these proteins.
• Haemoglobin: Respiratory protein found in RB Cells
• Cytochromes: These are the mitochondrial enzymes of the respiratory chain.
• Catalase: This is the enzyme that decomposes H2O2 to water and O2.
• Peroxidase: It is an oxidative enzyme.
(b) Others
• Flavoprotein: It is a cellular oxidation-reduction protein which has riboflavin a constituent of B complex
vitamin as its prosthetic group. This is yellow in colour.
• Visual purple: It is a protein of the retina in which the prosthetic group is a carotenoid pigment which is
purple in colour.
5. Phosphoproteins
These are the proteins with phosphoric acid as organic phosphate but not the phosphate containing substances
such as nucleic acids and phospholipids. (a) Casein and (b) Ovovitellin are the two important groups of
phosphoproteins found in milk, egg-yolk respectively.
They contain about 1 per cent of phosphorus. Similar proteins are stated to be present in fish eggs. They are
sparingly soluble in water, and very dilute acid in cold but readily soluble in very dilute alkali. The phosphoric
acid which is esterified through the –OH groups of serine and threonine is liberated from organic combination
by warming with NaOH and can only be detected by Ammonium Molybdate.
6. Lipoproteins
The lipoproteins are formed in combination with lipids as their prosthetic group (Refer chapter on Metabolism
of Lipids).
7. Metalloproteins
As the name indicates, they contain a metal ion as their prosthetic group. Several enzymes contain metallic
elements such as Fe, Co, Mn, Zn, Cu, Mg, etc.
Examples: Ferritin: Contains Fe, Carbonic Anhydrase: Contains Zn, Ceruloplasmin: Contains Cu.
C. DERIVED PROTEINS:
This class of proteins includes those protein products formed from the simple and conjugated proteins. It is
not a well-defined class of proteins. These are produced by various physical and chemical factors and are
divided in two major groups.
(a) Primary derived proteins: Denatured or coagulated proteins are placed in this group. Their molecular
weight is the same as native protein, but they differ in solubility, precipitation and crystallisation. Heat, Xray, UV rays, vigorous shaking, acid, alkali cause denaturation and give rise to primary derived proteins.
There is an intramolecular rearrangement leading to changes in their properties such as solubility. Primary
derived proteins are synonymous with denatured proteins in which peptide bonds remain intact.
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1. Proteans: These are insoluble products formed by the action of water, very dilute acids and enzymes. They
are predominantly formed from certain globulins.
Example: Myosan: From myosin, Edestan: From elastin and Fibrin: From fibrinogen.
2. Metaproteins: They are formed from further action of acids and alkalies on proteins. They are generally
soluble in dilute acids and alkalies but insoluble in neutral solvents, e.g. acid and alkali metaproteins.
3. Coagulated proteins: The coagulated proteins are insoluble products formed by the action of heat or
alcohol on native proteins.
Examples: include cooked meat, cooked egg albumin and alcohol precipitated proteins.
(b) Secondary derived proteins: These are the proteins formed by the progressive hydrolysis of proteins at
their peptide linkages. They represent a great complexity with respect to their size and amino acid
composition. They are roughly called as proteoses, peptones and peptides according to relative average
molecular size.
1. Proteoses or albumoses: These are the hydrolytic products of proteins which are soluble in water and are
coagulated by heat and are precipitated from their solution by saturation with Ammonium Sulphate.
2. Peptones: These are the hydrolytic products of proteoses. They are soluble in water, not coagulated by
heat and not precipitated by saturation with Ammonium sulphate. They can be precipitated by
phosphotungstic acid.
Examples: Protein products obtained by the enzymatic digestion of proteins.
3. Peptides: Peptides are composed of only a small number of amino acids joined as peptide bonds. They are
named according to the number of amino acids present in them.
• Dipeptides—made up of two amino acids,
• Tripeptides—made of three amino acid, etc. Peptides are water soluble and are not coagulated by heat, are
not salted out of solution and can be precipitated by phosphotungstic acid.
Hydrolysis: The complete hydrolytic decomposition of a protein generally follows the stages given below:
LEVELS OF PROTEINS STRUCTURE
There are four levels of structure in proteins: primary, secondary, tertiary and, sometimes but not always,
quaternary.
PRIMARY STRUCTURE
The primary level of structure in a protein is the linear sequence of amino acids as joined together by peptide
bonds. This sequence is determined by the sequence of nucleotide bases in the gene encoding the protein. Also
included under primary structure is the location of any other covalent bonds. These are primarily disulfide
bonds between cysteine residues that are adjacent in space but not in the linear amino acid sequence. These
covalent cross-links between separate polypeptide chains or between different parts of the same chain are
formed by the oxidation of the SH groups on cysteine residues.
SECONDARY STRUCTURE
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The secondary level of structure in a protein is the regular folding of regions of the polypeptide chain. The
two most common types of protein fold are the α-helix and the β-pleated sheet.
α-helix
In the rod-like α-helix, the amino acids arrange themselves in a regular helical conformation (Figure 2a). The
carbonyl oxygen of each peptide bond is hydrogen bonded to the hydrogen on the amino group of the fourth
amino acid away (Figure 2b), with the hydrogen bonds running nearly parallel to the axis of the helix. In αhelix there are 3.6 amino acids per turn of the helix covering a distance of 0.54 nm, and each amino acid
residue represents an advance of 0.15 nm along the axis of the helix. The side-chains of the amino acids are
all positioned along the outside of the cylindrical helix (Figure 2c).
β-pleated sheet
In the β-pleated sheet, hydrogen bonds form between the peptide bonds either in different polypeptide chains
or in different sections of the same polypeptide chain (Figure 3a). The planarity of the peptide bond forces
the polypeptide to be pleated with the side-chains of the amino acids protruding above and below the sheet
(Figure 3b). Adjacent polypeptide chains in β-pleated sheets can be either parallel or antiparallel depending
on whether they run in the same direction or in opposite directions, respectively (Figure 3c). The polypeptide
chain within a β-pleated sheet is fully extended, such that there is a distance of 0.35 nm from one Cα atom to
the next. β-Pleated sheets are always slightly curved and, if several polypeptides are involved, the sheet can
close up to form a β-barrel. Multiple β-pleated sheets provide strength and rigidity in many structural
proteins, such as silk fibroin, which consists almost entirely of stacks of antiparallel β-pleated sheets.
Figure 2: The folding of the polypeptide chain into an α-helix. (a) Model of an α-helix with only the Cα atoms along the
backbone shown; (b) in the α-helix the CO group of residue n is hydrogen bonded to the NH group on residue (n + 4); (c) crosssectional view of an α-helix showing the positions of the side-chains (R groups) of the amino acids on the outside of the helix.
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Figure 3: The folding of the polypeptide chain in a β-pleated sheet. (a) Hydrogen bonding between two sections
of a polypeptide chain forming a β-pleated sheet; (b) a side-view of one of the polypeptide chains in a β-pleated sheet showing the
side-chains (R groups) attached to the Cα atoms protruding above and below the sheet; (c) because the polypeptide chain has
polarity, either parallel or antiparallel β-pleated sheets can form.
In order to fold tightly into the compact shape of a globular protein, the polypeptide chain often reverses
direction, making a hairpin or β-turn. In these β-turns the carbonyl oxygen of one amino acid is hydrogen
bonded to the hydrogen on the amino group of the fourth amino acid along (Figure 4).
Figure 4: The folding of the polypeptide chain in a β-turn.
TERTIARY STRUCTURE
The third level of structure found in proteins, tertiary structure, refers to the spatial arrangement of amino
acids that are far apart in the linear sequence as well as those residues that are adjacent. It is the sequence of
amino acids that specifies this final three-dimensional structure (Figure 5). The polypeptide chain folds
spontaneously so that the majority of its hydrophobic side-chains are buried in the interior, and the majority
of its polar, charged side-chains are on the surface.
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Once folded, the three-dimensional biologically-active (native) conformation of the protein is maintained
not only by hydrophobic interactions, but also by electrostatic forces, hydrogen bonding and, if present,
the covalent disulphide bonds. The electrostatic forces include salt bridges between oppositely charged
groups and the multiple weak van der Waals interactions between the tightly packed aliphatic side-chains in
the interior of the protein.
Figure 5: The four levels of structure in proteins. (a) Primary structure (amino acid sequence), (b)
secondary structure (α-helix), (c) tertiary structure, (d) quaternary structure.
QUATERNARY STRUCTURE
Proteins containing more than one polypeptide chain, such as hemoglobin, exhibit a fourth level of protein
structure called quaternary structure (Figure 5). This level of structure refers to the spatial arrangement of the
polypeptide subunits and the nature of the interactions between them. These interactions may be covalent links
(e.g. disulfide bonds) or noncovalent interactions (electrostatic forces, hydrogen bonding, hydrophobic
interactions).
PROTEIN STABILITY
The three-dimensional conformation of a protein is maintained by a range of noncovalent interactions
(electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulfide bonds) in
addition to the peptide bonds between individual amino acids.
ELECTROSTATIC FORCES
Electrostatic forces include the interactions between two ionic groups of opposite charge, for example the
ammonium group of Lys and the carboxyl group of Asp, often referred to as an ion pair or salt bridge. In
addition, the noncovalent associations between electrically neutral molecules, collectively referred to as van
der Waals forces, arise from electrostatic interactions between permanent and/or induced dipoles, such as the
carbonyl group in peptide bonds. These are very short-range interactions between atoms that occur when atoms
are packed very closely to each other.
VAN DER WAALS FORCES/ INTERACTIONS
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These forces arise from electrostatic attraction between the positively charged nucleus of one atom and the
negatively charged electrons of the other. Both attractive forces and repulsive forces are included in van der
Waals interactions. The attractive forces are due primarily to instantaneous dipole-induced dipole interactions
that arise because of fluctuations in the electron charge distributions of adjacent nonbonded atoms. Individual
van der Waals interactions are weak ones (with stabilization energies of 4.0 to 1.2 kJ/mol), but many such
interactions occur in a typical protein, and, by sheer force of numbers, they can represent a significant
contribution to the stability of a protein.
HYDROGEN BONDS
Hydrogen bonding means sharing a hydrogen atom between one atom that has a hydrogen atom (donor) and
another atom that has a lone pair of electrons (acceptor). They are predominantly electrostatic interactions
between a weakly acidic donor group and an acceptor atom that bears a lone pair of electrons, which thus has
a partial negative charge that attracts the hydrogen atom. In biological systems the donor group is an oxygen
or nitrogen atom that has a covalently attached hydrogen atom, and the acceptor is either oxygen or
nitrogen (Figure 1). Hydrogen bonds are normally in the range 0.27–0.31 nm and are highly directional, i.e.
the donor, hydrogen and acceptor atoms are collinear. Hydrogen bonds are stronger than van der Waals forces
but much weaker than covalent bonds.
Figure 1: Examples of hydrogen bonds (shown as dotted lines).
HYDROPHOBIC FORCES
The hydrophobic effect is the name given to those forces that cause nonpolar molecules to minimize their
contact with water. This is clearly seen with amphipathic molecules such as lipids and detergents which form
micelles in aqueous solution. Proteins, too, find a conformation in which their nonpolar side-chains are largely
out of contact with the aqueous solvent, and thus hydrophobic forces are an important determinant of protein
structure, folding and stability. Proteins fold in order to put as much of the hydrophobic part out of contact
with water as possible. In proteins, the effects of hydrophobic forces are often termed hydrophobic bonding,
to indicate the specific nature of protein folding under the influence of the hydrophobic effect. For protein
folding, the hydrophobic interaction that provides most of the driving force. As water squeezes out the
hydrophobic side chains, distant parts of the protein are brought together into a compact structure. The
hydrophobic core of most globular proteins is very compact, and the pieces of the hydrophobic core must fit
together rather precisely.
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Figure 2: The Hydrophobic Interaction
DISULFIDE BONDS
Disulfide bonds are covalent bonds form between Cys residues that are close together in the final
conformation of the protein and function to stabilize its three-dimensional structure. Disulfide bonds are really
only formed in the oxidizing environment of the endoplasmic reticulum, and thus are found primarily in
extracellular and secreted proteins.
Figure 3: Formation of a disulfide bond between two cysteine residues
PROTEIN DENATURATION
Protein denaturation a process in which the three-dimensional conformation of a protein is changed from its
native state without rupture of peptide bonds. It may include disulphide bond rupture or chemical modification
of certain groups in the protein if these processes are also accompanied by changes in its overall threedimensional structure. Denaturation is frequently irreversible and accompanied by loss of solubility
(especially at the isoelectric point) and/or of biological activity. Denaturation can be caused by heating,
changes in pH, or exposure to certain chemicals.
1. When a protein in solution is heated, its conformationally sensitive properties, such as optical rotation,
viscosity, and UV absorption, change abruptly over a narrow temperature range.
2. pH variations alter the ionization states of amino acid side chains which changes protein charge
distributions and H bonding requirements.
3. Detergents significantly perturb protein structures at concentrations as low as 10-6 M. They hydrophobically
associate with the nonpolar residues of a protein, thereby interfering with the hydrophobic interactions
responsible for the protein’s native structure.
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4. High concentrations of water-soluble organic substances, such as aliphatic alcohols, interfere with the
hydrophobic forces stabilizing protein structures through their own hydrophobic interactions with water.
5. Some salts, such as (NH4)2SO4 and KH2PO4, stabilize the native protein structure; others, such as KCl and
NaCl, have little effect; and yet others, such as KSCN and LiBr, destabilize it. The order of effectiveness of
the various ions in stabilizing a protein, which is largely independent of the identity of the protein, parallels
their capacity to salt out proteins. This order is known as the Hofmeister series:
The ions in the Hofmeister series that tend to denature proteins, I‾, ClO4‾, SCN‾, Li+, Mg2+, Ca2+, and Ba2+,
are said to be chaotropic. In addition to this list is guanidinium ion (Gu+) and the nonionic urea, which, in
concentrations in the range 5 to 10 M, are the most commonly used protein denaturants. Chaotropic agents
disrupt hydrophobic interactions in proteins.
PROTEIN RENATURATION
Renaturation of protein refers to the reconstruction of the original conformation of a protein following
denaturation. A good example is the renaturation of RNase A. RNase A, a 124-residue single chain protein,
is completely unfolded and its four disulphide bonds reductively cleaved in an 8M urea solution containing 2mercaptoethanol. Yet dialyzing away the urea and exposing the resulting solution to O2 at pH 8 yields a protein
that is virtually 100% enzymatically active and physically indistinguishable from native RNase A. The protein
must therefore have spontaneously renatured.
REFERENCES AND FURTHER READINGS
1.
Moran LA, Horton HR, Scrimgeour, KG, and Perry MD (2012) Principles of Biochemistry 5th Edn,
Pearson Education, Inc., Boston, USA.
2.
Rodwell VW, Bender DA, Botham KM, Kennelly PJ, and Weil PA. (2015) Harper’s Illustrated
Biochemistry 30th Edn, The McGraw-Hill Companies, Inc. New York, USA
3.
Voet D and Voet JG (2011) Biochemistry 4th Edn John Wiley & Sons, Inc., Hoboken, NJ USA.
4.
Garrett, R. H. and Grisham, C. M. (2013) Biochemistry 5th Edn Brooks/Cole, Belmont, USA
5.
Biochemistry, 7th Edition, Mary K. Campbell, Shawn O. Farrell, Brooks/Cole
20 Davis Drive, Belmont, CA 94002-3098, USA
6.
Chatterjea MN and Shinde R (2012) Textbook of Medical Biochemistry 8th Edn Jaypee Brothers
Medical Publishers (P) Ltd, New Delhi, India.
7.
Bhagavan, N. V. and Ha, C. (2015) Essentials of Medical Biochemistry: With Clinical Cases, Second
Edition, Elsevier Inc, San Diego, USA
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