This document provides an overview of biochemistry, including its definition, objectives, and relationships to other life sciences. It discusses that proteins are the most abundant biomolecules in cells, consisting of linear polymers of 20 amino acids. The major techniques used in biochemistry to study cellular components and reactions are also summarized.

1. Proteins
What is Biochemistry?
Biochemistry can be defined as the science concerned with the chemical basis of life
(Gk bios “life”). The cell is the structural unit of living systems. Thus, biochemistry can
also be described as the science concerned with the chemical constituents of living cells
and with the reactions and processes they undergo. By this definition, biochemistry
encompasses large areas of cell biology, of molecular biology, and of molecular genetics.
The Aim of Biochemistry Is to Describe & Explain in Molecular Terms, All Chemical
Processes of Living Cells:
The major objective of biochemistry is the complete understanding, at the molecular level, of all
of the chemical processes associated with living cells. To achieve this objective, biochemists have
sought to isolate the numerous molecules found in cells, determine their structures, and analyze how
they function. Many techniques have been used for these purposes; some of them are summarized in
Table 1.
Knowledge of Biochemistry Is Essential to All Life Sciences:
The biochemistry of the nucleic acids lies at the heart of genetics; in turn, the use of genetic
approaches has been critical for elucidating many areas of biochemistry. Physiology, the study of
body function, overlaps with biochemistry almost completely. Immunology employs numerous
biochemical techniques, and many immunologic approaches have found wide use by biochemists.
Pharmacology and pharmacy rest on a sound knowledge of biochemistry and physiology; in
particular, most drugs are metabolized by enzyme-catalyzed reactions. Poisons act on biochemical
reactions or processes; this is the subject matter of toxicology. Biochemical approaches are being
used increasingly to study basic aspects of pathology (the study of disease), such as inflammation,
cell injury, and cancer. Many workers in microbiology, zoology, and botany employ biochemical
approaches almost.
Table 1: The principal methods and preparations used in biochemical laboratories.
Methods for separating and purifying biomolecules:
Salt fractionation (e.g., precipitation of proteins with ammonium sulfate).
Chromatography: paper; ion exchange; affinity; thin-layer; gas-liquid; high-pressure
liquid; gel filtration.
Electrophoresis: paper; high-voltage; agarose; cellulose acetate; starch gel;
polyacrylamide gel; SDS-polyacrylamide gel.
Ultracentrifugation
Methods for Determining Biomolecular Structures:
Elemental analysis.
UV, visible, infrared, and NMR spectroscopy.
Use of acid or alkaline hydrolysis to degrade the biomolecule under study into its basic
constituents.
Use of a battery of enzymes of known specificity to degrade the biomolecule under
study (e.g., proteases, nucleases, glycosidases).
Mass spectrometry.
Specific sequencing methods (e.g., for proteins and nucleic acids).
X-ray crystallography.
Preparations for Studying Biochemical Processes:
Whole animal (includes transgenic animals and animals with gene knockouts).

2. 1
Isolated perfused organ.
Tissue slice.
Whole cells.
Homogenate.
Isolated cell organelles.
Sub-fractionation of organelles.
Purified metabolites and enzymes.
Isolated genes (including polymerase chain reaction and site-directed mutagenesis)
1Most of these methods are suitable for analyzing the components present in cell homogenates and
other biochemical preparations. The sequential use of several techniques will generally permit
purification of most biomolecule. The reader is referred to texts on methods of biochemical research
for details. These relationships are not surprising, because life as we know it depends on biochemical
reactions and processes. In fact, the old barriers among the life sciences are breaking down, and
biochemistry is increasingly becoming their common language.
A Reciprocal Relationship between Biochemistry& Medicine Has Stimulated Mutual
Advances
The two major concerns for workers in the health sciences and particularly physicians are the
understanding and maintenance of health and the understanding and effective treatment of diseases.
Biochemistry impacts enormously on both of these fundamental concerns of medicine. In fact, the
interrelationship of biochemistry and medicine is a wide, two- way street.
Biochemical studies have illuminated many aspects of health and disease, and conversely, the
study of various aspects of health and disease has opened up new areas of biochemistry. Some
examples of this two-way street are shown in Figure 1. For instance, knowledge of protein structure
and function was necessary to elucidate the single biochemical difference between normal
hemoglobin and sickle cell hemoglobin. On the other hand, analysis of sickle cell hemoglobin has
contributed significantly to our understanding of the structure and function of both normal
hemoglobin and other proteins. Analogous examples of reciprocal benefit between biochemistry and
medicine could be cited for the other paired items shown in Figure 1. Another example is the
pioneering work of Archibald Garrod, a physician in England during the early 1900s. He studied
patients with a number of relatively rare disorders (alkaptonuria, albinism, cystinuria, and pentosuria;
these are described) and established that these conditions were genetically determined. Garrod
designated these conditions as inborn errors of metabolism.
Figure 1: Examples of the two-way street connecting biochemistry and medicine. Knowledge of the
biochemical molecules shown in the top part of the diagram has clarified our understanding of the
diseases shown in the bottom half and conversely, analyses of the diseases shown below have cast
light on many areas of biochemistry. Note that sickle cell anemia is a genetic disease and that both
atherosclerosis and diabetes mellitus have genetic components.
His insights provided a major foundation for the development of the field of human biochemical
genetics. More recent efforts to understand the basis of the genetic disease were known as familial
hypercholesterolemia, which results in severe atherosclerosis at an early age, have led to dramatic

3. 2
progress in understanding of cell receptors and of mechanisms of uptake of cholesterol into cells.
Studies of oncogenes in cancer cells have directed attention to the molecular mechanisms involved
in the control of normal cell growth. These and many other examples emphasize how the study of
disease can open up areas of cell function for basic biochemical research.
The relationship between medicine and biochemistry has important implications for the former.
As long as medical treatment is firmly grounded in knowledge of biochemistry and other basic
sciences, the practice of medicine will have a rational basis that can be adapted to accommodate new
knowledge. This contrasts with unorthodox health cults and at least some “alternative medicine”
practices, which are often founded on little more than myth and wishful thinking and generally lack
any intellectual basis.
Normal biochemical processes are the basis of health:
The World Health Organization (WHO) defines health as a state of “complete physical, mental
and social well-being and not merely the absence of disease and infirmity.” From a strictly
biochemical viewpoint, health may be considered that situation in which all of the many thousands
of intra- and extracellular reactions that occur in the body are proceeding at rates commensurate with
the organism’s maximal survival in the physiologic state. However, this is an extremely reductionist
view, and it should be apparent that caring for the health of patients requires not only a wide
knowledge of biologic principles but also of psychologic and social principles.
Biochemical Research Has Impact on Nutrition & Preventive Medicine:
One major prerequisite for the maintenance of health is that there is optimal dietary intake of a
number of chemicals; the chief of these are vitamins, certain amino acids, certain fatty acids, various
minerals, and water. Because much of the subject matter of both biochemistry and nutrition is
concerned with the study of various aspects of these chemicals, there is a close relationship between
these two sciences. Moreover, more emphasis is being placed on systematic attempts to maintain
health and forestall disease, i.e., on preventive medicine. Thus, nutritional approaches to for example,
the prevention of atherosclerosis and cancer are receiving increased emphasis. Understanding
nutrition depends to a great extent on knowledge of biochemistry.
Most & Perhaps All Disease Has a Biochemical Basis:
We believe that most if not all diseases are manifestations of abnormalities of molecules, chemical
reactions, or biochemical processes. The major factors responsible for causing diseases in animals
and humans are listed in Table 2. All of them affect one or more critical chemical reactions or
molecules in the body. Numerous examples of the biochemical bases of diseases will be encountered
in this text; the majority of them are due to causes 5, 7, and 8. In most of these conditions, biochemical
studies contribute to both the diagnosis and treatment. Some major uses of biochemical investigations
and of laboratory tests in relation to diseases are summarized in Table 3. Additional examples of
many of these uses are presented in various sections of this text.
Impact of the Human Genome Project (HGP) on Biochemistry & Medicine:
Remarkable progress was made in the late 1990s in sequencing the human genome. This
culminated in July 2000, when leaders of the two groups involved in this effort (the International
Human Genome Sequencing Consortium and Celera Genomics, a private company) announced that
over 90% of the genome had been sequenced. Draft versions of the sequence were published in early
2001. It is anticipated that the entire sequence will be completed by 2003. The implications of this
work for biochemistry, all of biology, and for medicine are tremendous, and only a few points are
mentioned here. Many previously unknown genes have been revealed; their protein products await
characterization. New light has been thrown on human evolution, and procedures for tracking disease

4. 3
genes have been greatly refined. The results are having major effects on areas such as proteomics,
bioinformatics, biotechnology, and pharmacogenomics.
Table 2: The major causes of diseases. The entire causes listed act by influencing the various
biochemical mechanisms in the cell or in the body.*
1) Physical agents: mechanical trauma, extremes of temperature, sudden changes in
atmospheric pressure, radiation, electric shock.
2) Chemical agents, including drugs: certain toxic compounds, therapeutic drugs, etc.
3) Biologic agents: viruses, bacteria, fungi, higher forms of parasites.
4) Oxygen lack: loss of blood supply, depletion of the oxygen-carrying capacity of the
blood, poisoning of the oxidative enzymes.
5) Genetic disorders: congenital, molecular.
6) Immunologic reactions: anaphylaxis, autoimmune disease.
7) Nutritional imbalances: deficiencies, excesses.
8) Endocrine imbalances: hormonal deficiencies, excesses.
*Adapted, with permission, from Robbins el al., The Pathologic Basis of Disease, 3rd ed.
Saunders, 1984.
Table 3: Some uses of biochemical investigations and laboratory tests in relation to diseases.
Use Example
1. To reveal the fundamental causes and
mechanisms of diseases
Demonstration of the nature of the genetic
defects in cystic fibrosis.
2. To suggest rational treatments of
diseases based on (1) above
A diet low in phenylalanine for treatment of
phenyl ketonuria.
3. To assist in the diagnosis of specific
diseases MB
Use of the plasma enzyme creatine kinase
(CK-MB) in the diagnosis of myocardial
infarction.
4. To act as screening tests for the early
diagnosis of certain diseases
Use of measurement of blood thyroxine or
thyroid-stimulating hormone (TSH) in the
neonatal diagnosis of congenital
hypothyroidism.
5. To assist in monitoring the progress
(e.g., recovery, worsening, remission,
or relapse) of certain diseases.
Use of the plasma enzyme alanine
aminotransferase (ALT) in monitoring the
progress of infectious hepatitis
6. To assist in assessing the response of
diseases to therapy
Use of measurement of blood
carcinoembryonic antigen (CEA) in certain
patients who have been treated for cancer of
the colon & HbC1.
Which are the most abundant biomolecules in cells?
Proteins are the most abundant biological macromolecules, occurring in all cells and all parts of
cells and constitute 50% of the dry weight of most organisms. Hence the name given, protein = first
or foremost. Proteins also occur in great variety; thousands of different kinds, ranging in size from
relatively small peptides to huge polymers with molecular weights in the millions, may be found in a
single cell. Moreover, proteins exhibit enormous diversity of biological function and are the most
important final products of the information pathways. Virtually every life process depends on this
class of molecules. For example, enzymes and polypeptide hormones direct and regulate metabolism
in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen
forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in

5. 4
reinforced concrete. In the bloodstream, proteins, such as hemoglobin and plasma albumin, shuttle
molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. Moreover,
proteins are the molecular instruments through which genetic information is expressed.
Relatively simple monomeric subunits provide the key to the structure of the thousands of different
proteins. All proteins, whether from the most ancient lines of bacteria or from the most complex forms
of life, are constructed from the same ubiquitous set of 20 amino acids, covalently linked in
characteristic linear sequences. In short, proteins display an incredible diversity of functions, yet all
share the common structural feature of being linear polymers of amino acids. Proteins may be defined
as compounds of high M.W. range from 5000 – 25,000,000 and consists of α-amino acids.
Amino Acids: Building Blocks of proteins
Structure of a typical Amino Acid:
Although, over 300 amino acids occur in nature, only 20 occur in proteins. Amino acids contain
both amino (NH2) and carboxylic (COOH) functional groups. Amino acids found in proteins (except
proline, an imino acid) are α-amino acids, i.e. both amino and carboxyl groups are present on the α-
carbon.
Figure 2 -
amino acids. (Proline, a cyclic amino acid, is the exception). The R group or side chain (red) attached
to the -carbon (blue) is different in each amino acid.
Two conventions are used to identify the carbons in an amino acid. The additional carbons in an
R group are commonly designated  and so forth, proceeding out from the -carbon. For most
other organic molecules, carbon atoms are simply numbered from one end, giving highest priority
(C-1) to the carbon with the substituents containing the atom of highest atomic number. Within this
latter convention, the carboxyl carbon of an amino acid would be C-1 and the - carbon would be C-
2. In some cases, such as amino acids with heterocyclic R groups, the Greek lettering system is
ambiguous and the numbering convention is therefore used.
The α-carbon of all amino acids found in proteins (with the exception of glycine) has four
different groups substituted on it; a carboxyl group, an amino group, an R group and a hydrogen atom.
The α-carbon is thus asymmetric and such compounds exist in two different isomeric forms, which
are identical in all chemical and physical properties except one, the direction in which they cause the
rotation of plane polarized light in a polarimeter. Amino acids are thus referred to as being optically
active, meaning that they can rotate plane polarized light in one direction (right) or the other (left).
Those that rotate plane polarized light to the right (clockwise) are called dextrorotatory isomer,
designated as (+) and those to the left (counter clockwise) are called levorotatory isomer, designated
as (-).
Except for glycine, which has no asymmetric carbon atom, the amino acids present in naturally
occurring protein molecules are the L-isomers. Some D-amino acids do occur in living matter (e.g.
D-phenylalanine in Gramicidin S, an antibiotic) but they have not been found in proteins, which are
biosynthesized on the ribosome. Polypeptides containing non-protein amino acids are not synthesized
on the ribosome.



6. 5
Amino Acids Share Common Structural Features:
All 20 of the common amino acids are -amino acids. They differ from each other in their side
chains, or R groups, which vary in structure, size, and electric charge, and which influence the
solubility of the amino acids in water. In addition to these 20 amino acids there are many less common
ones. Some are residues modified after a protein has been synthesized; others are amino acids present
in living organisms but not as constituents of proteins. The common amino acids of proteins have
been assigned three-letter abbreviations and one-letter symbols (Table 4), which are used as shorthand
to indicate the composition and sequence of amino acids polymerized in proteins.
-carbonis bonded to four different groups:
a carboxyl group, an amino group, an R group, and a hydrogen atom (Fig. 2; in glycine, the R group
is another hydrogen atom). The -carbon atom is thus a chiral center. Because of the tetrahedral
arrangement of t α-carbon atom, the four different groups can occupy
two unique spatial arrangements, and thus amino acids have two possible stereo isomers. Since they
are non-super imposable mirror images of each other (Fig. 3), the two forms represent a class of stereo
isomers called enantiomers. All molecules with a chiral center are also optically active, they rotate
plane of polarized light.
Special nomenclature has been developed to specify the absolute configuration of the four
substituents of asymmetric carbon atoms. The absolute configurations of simple sugars and amino
acids are specified by the D, L system (Fig. 3), based on the absolute configuration of the three-carbon
sugar glyceraldehyde, a convention proposed by Emil Fischer in 1891.

7. 6
Figure 3: Stereoisomerism in α-amino acids.
The two stereoisomers of alanine, L- and D-alanine, are non-super imposable mirror images of
each other (enantiomers).
(b, c), Two different conventions for showing the configurations in space of stereo isomers. In
perspective formulas (b) the solid wedge-shaped bonds project out of the plane of the paper, the
dashed bonds behind it. In projection formulas (c) the horizontal bonds are assumed to project out of
the plane of the paper, the vertical bonds behind. However, projection formulas are often used
casually and are not always intended to portray a specific stereo chemical configuration.
Common Amino Acids:
Amino Acids can be classified by R Group:
Knowledge of the chemical properties of the common amino acids is central to an understanding
of biochemistry. The topic can be simplified by grouping the amino acids into five main classes based
on the properties of their R groups (Fig. 4), in particular, their polarity, or tendency to interact with
water at biological pH (near pH 7.0). The polarity of the R groups varies widely, from nonpolar and
hydrophobic (water-insoluble) to highly polar and hydrophilic (water-soluble).
The structures of the 20 common amino acids are shown in Figure 3. Within each class there are
gradations of polarity, size, and shape of the R groups.
 Nonpolar, Aliphatic R Groups: The R groups in this class of amino acids are nonpolar and
hydrophobic. The side chains of alanine, valine, leucine, and isoleucine tend to cluster together
within proteins, stabilizing protein structure by means of hydrophobic interactions. Glycine has
the simplest structure. Although it is formally nonpolar, its very small side chain makes no real
contribution to hydrophobic interactions. Methionine, one of the two sulfur-containing amino
acids, has a nonpolar thioether group in its side chain. Proline has an aliphatic side chain with a
distinctive cyclic structure. The secondary amino (imino) group of proline residues is held in a
rigid conformation that reduces the structural flexibility of polypeptide regions containing
proline.
 Aromatic R Groups: Phenylalanine, tyrosine, and tryptophan, with their aromatic side chains, are
relatively nonpolar (hydrophobic). All can participate in hydrophobic interactions. The hydroxyl
group of tyrosine can form hydrogen bonds, and it is an important functional group in some
enzymes. Tyrosine and tryptophan are significantly more-polar than phenylalanine, because of
the tyrosine hydroxyl group and the nitrogen of the tryptophan indole ring. Tryptophan and
tyrosine, and to a much lesser extent phenylalanine, absorb ultraviolet light. This accounts for
the characteristic strong absorbance of light by most proteins at a wavelength of 280 nm, a
property exploited by researchers in the characterization of proteins.

8. 7
The structural formulas show the state of ionization that would predominate at pH 7.0. The
unshaded portions are those common to all the amino acids; the portions shaded in red are the R
groups. Although the R group of histidine is shown uncharged, its pKa is such that a small but
significant fraction of these groups are positively charged at pH 7.0.
 Polar, Uncharged R Groups: The R groups of these amino acids are more soluble in water, or
more hydrophilic, than those of the nonpolar amino acids, because they contain functional groups
that form hydrogen bonds with water. This class of amino acids includes serine, threonine,
cysteine, asparagine, and glutamine. The polarity of serine and threonine is contributed by their
hydroxyl groups; that of cysteine by its sulfhydryl group; and that of asparagine and glutamine by
their amide groups.
Asparagine and glutamine are the amides of two other amino acids also found in proteins,
aspartate and glutamate, respectively, to which asparagine and glutamine are easily hydrolyzed by
acid or base. Cysteine is readily oxidized to form a covalently linked dimeric amino acid called
cystine, in which two cysteine molecules or residues are joined by a disulfide bond. The disulfide-
linked residues are strongly hydrophobic (nonpolar). Disulfide bonds play a special role in the
structures of many proteins by forming covalent links between parts of a protein molecule or
between two different polypeptide chains.
 Positively Charged (Basic) R Groups: Three of the common amino acids have side chains with
net positive charges at neutral pH: histidine, arginine, and lysine. The ionized group of histidine
is an imidazolium, while that of arginine is a guanidinium, and lysine contains a protonated alkyl
amino group. The side chains of the latter two amino acids are fully protonated at pH 7, but
histidine, with a side chain pKa of 6.0 is only 10% protonated at pH 7. with a pKa near neutrality,
histidine side chains play important roles as proton donors and acceptors in many enzyme
reactions. Histidine-containing peptides are important biological buffers. Arginine and lysine side
chains, which are protonated under physiological conditions, participate in electrostatic
interactions in proteins. In many enzyme-catalyzed reactions, a His residue facilitates the reaction
by serving as a proton donor/acceptor.
 Negatively Charged (Acidic) R Groups: There are two acidic amino acids: aspartic acid and
glutamic acid, whose R groups contain a carboxyl group. These side chain carboxyl groups are
weaker acids than the α-COOH group, but are sufficiently acidic to exist as -COO- at neutral pH.
Aspartic acid and glutamic acid thus have a net negative charge at pH 7. These negatively charged
amino acids play several important roles in proteins. Many proteins that bind metal ions for
structural or functional purposes possess metal binding sites containing one or more aspartate and
glutamate side chains. Carboxyl groups may also act as nucleophiles in certain enzyme reactions
and may participate in a variety of electrostatic bonding interaction.

9. 8
Figure 5: Reversible formation of a disulfide bond by the oxidation of two molecules of cysteine.
Disulfide bonds between Cys residues stabilize the structures of many proteins.
Uncommon Amino Acids Also Have Important Functions:
 In addition to the 20 common amino acids, proteins may contain residues created by modification
of common residues already incorporated into a polypeptide (Fig. 6a). Among these uncommon
amino acids are 4-hydroxyproline, a derivative of proline, and 5-hydroxylysine, derived from
lysine. The former is found in plant cell wall proteins, and both are found in collagen, a fibrous
protein of connective tissues. 6-N Methyl-lysine is a constituent of myosin, a contractile protein
of muscle. Another important uncommon amino acid is -carboxyglutamate, found in the blood
clotting protein prothrombin and in certain other proteins that bind Ca2+ as part of their biological
function.
 More complex is desmosine, a derivative of four Lys residues, which is found in the fibrous protein
elastin. Selenocysteine is a special case. This rare amino acid residue is introduced during protein
synthesis rather than created through a post synthetic modification. It contains selenium rather than
the sulfur of cysteine. Actually derived from serine, selenocysteine is a constituent of just a few
known proteins. Some 300 additional amino acids have been found in cells. They have a variety
of functions but are not constituents of proteins. Ornithine and citrulline (Fig. 6b) deserve special
note because they are key intermediates (metabolites) in the biosynthesis of arginine and in the
urea cycle.
 These amino acids are formed as a result of modification of the parent amino acid after the protein
chain is biosynthesized.

10. 9
Figure 6: Uncommon Amino Acids.
 Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra
functional groups added by modification reactions are shown in red. Desmosine is formed from
four Lys residues (the four carbon backbones are shaded in yellow). Note the use of either numbers
or Greek letters to identify the carbon atoms in these structures.
 Ornithine and citrulline, which are not found in proteins, are intermediates in the biosynthesis of
arginine and in the urea cycle.
Amino Acids Not Found in Proteins:
Certain amino acids and their derivatives, while not found in proteins, nonetheless are
biochemically important. -Amino butyric acid, or GABA, is produced by the decarboxylation of
glutamic acid and is a potent neurotransmitter. Histamine, which is synthesized by decarboxylation
of histidine, and serotonin, which is derived from tryptophan, similarly functions as neurotransmitters
and regulators. -Alanine is bound in nature in the peptides carnosine and anserine and is a component
of pantothenic acid (a vitamin), which comprises part of coenzyme A. Epinephrine (also known
adrenaline), derived from tyrosine, is an important hormone. Penicillamine is a constituent of the
penicillin antibiotics. Ornithine, betaine, homocysteine, and homoserine are important metabolic
intermediates. Citrulline is the immediate precursor of arginine.

11. 10
-Carboxyglutamic acid Phosphoserine Phosphothreonine
Phosphotyrosine -Alanine Histamine Serotonin
Amino Acids can Act as Acids and Bases:
Figure 7: Nonionic and Zwitter ionic forms of amino acids. The nonionic form does not occur in
significant amounts in aqueous solutions. The Zwitter ion predominates at neutral pH.
When an amino acid is dissolved in water, it exists in solution as the dipolar ion, or Zwitter ion
(German for “hybrid ion”), shown in Figure 7. A Zwitter ion can act as either an acid (proton donor):
Or a base (proton acceptor):

12. 11
Substances having this dual nature are amphoteric and are often called ampholytes (from
“amphoteric electrolytes”). A simple monoamino monocarboxylic -amino acid, such as alanine, is
a diprotic acid when fully protonated it has two groups, the -COOH group and the -NH3 group that
can yield protons:
Although Zwitter ions have opposite charges at there, two "poles" these are electrically neutral and
do not move in the electric field. Of the polar amino acids, Asp and Glu exist as -vely charged ions
while Lys, Arg and His exist as +vely charged ions at neutral pH. The polar amino acid exists as
Zwitter ions at their respective isoelectric points.
Isoelectric point of an amino acid:
The pH at which an amino acid exists completely in its zwitter ion state with no net electric charge
on it is isoelectric pH or isoelectric point. Molecules of an amino acid at this pH will not move in an
electric field.
Peptides and Proteins:
We now turn to polymers of amino acids, the peptides and proteins. Biologically occurring
polypeptides range in size from small to very large, consisting of two or three to thousands of linked
amino acid residues. Our focus is on the fundamental chemical properties of these polymers.
Peptides are Chains of Amino Acids:
Two amino acid molecules can be covalently joined through a substituted amide linkage, termed
a peptide bond, to yield a dipeptide. Such a linkage is formed by removal of the elements of water
(dehydration) from the -carboxyl group of one amino acid and the -amino group of another
(Fig. 8). Peptide bond formation is an example of a condensation reaction, a common class of
reactions in living cells. Under standard biochemical conditions, the equilibrium for the reaction
shown in Figure 9 favors the amino acids over the dipeptide. To make the reaction thermodynamically
more favorable, the carboxyl group must be chemically modified or activated so that the hydroxyl
group can be more readily eliminated. A chemical approach to this problem is outlined later in this
chapter.
Three amino acids can be joined by two peptide bonds to form a tripeptide; similarly, amino acids
can be linked to form tetrapeptides, pentapeptides, and so forth. When a few amino acids are joined
in this fashion, the structure is called an oligopeptide. When many amino acids are joined, the product
is called a polypeptide. Proteins may have thousands of amino acid residues. Although the terms
“protein” and “polypeptide” are sometimes used interchangeably, molecules referred to as
polypeptides generally have molecular weights below 10,000, and those called proteins have higher
molecular weights. Figure 8 shows the structure of a pentapeptide. As already noted, an amino acid
unit in a peptide is often called a residue (the part left over after losing a hydrogen atom from its
amino group and the hydroxyl moiety from its carboxyl group). In a peptide, the amino acid residue

13. 12
at the end with a free -amino group is the amino-terminal (or N-terminal) residue; the residue at the
other end, which has a free carboxyl group, is the carboxyl terminal (C-terminal) residue.
Although hydrolysis of a peptide bond is an exergonic reaction, it occurs slowly because of its
high activation energy. As a result, the peptide bonds in proteins are quite stable, with an average
half-life (t1/2) of about 7 years under most intracellular conditions.
Figure 8: Formation of a peptide bond by condensation.
The -amino group of one amino acid (with R2 group) acts as a nucleophile to displace the
hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow).
Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily
displaced. At physiological pH, the reaction shown does not occur to any appreciable extent.
Figure 9: The pentapeptide serylglycyltyrosylalanylleucine, or Ser–Gly–Tyr–Ala–Leu. Peptides are
named beginning with the aminoterminal residue, which by convention is placed at the left. The
peptide bonds are shaded in yellow; the R groups are in red.
 Characteristics of the peptide bond: The peptide bond has a partial double-bond character that
is, it is shorter than a single bond, and is rigid and planar (Figure 10). This prevents free rotation
around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the
bonds between the α-carbons and the α-amino or α-carboxyl groups can be freely rotated
(although they are limited by the size and character of the R-groups). This allows the polypeptide
chain to assume a variety of possible configurations. The peptide bond is generally a trans bond
(instead of cis, see Figure 10), in large part because of steric interference of the R-groups when
in the cis position.

14. 13
 Polarity of the peptide bond: Like all amide linkages, the -C= 0 and -N H groups of the peptide
bond are uncharged, and neither accept nor release protons over the pH range of 2 to 12. Thus,
the charged groups present in polypeptides consist solely of the N-terminal α-amino group, the
C-terminal α-carboxyl group, and any ionized groups present in the side chains of the constituent
amino acids. [Note: The -C= 0 and -N H groups of the peptide bond are polar, and are involved
in hydrogen bonds, for example, in α-helices and β-sheet structures.
Figure 10: Characteristics of the peptide bond.
Peptides can be distinguished by their Ionization Behavior:
Peptides contain only one free -amino group and one free -carboxyl group, at opposite ends of
the chain (Fig. 11). These groups ionize as they do in free amino acids, although the ionization
constants are different because an oppositely charged group is no longer linked to the -carbon. The
-amino and -carboxyl groups of all non-terminal amino acids are covalently joined in the peptide
bonds, which do not ionize and thus do not contribute to the total acid-base behavior of peptides.
However, the Rgroups of some amino acids can ionize, and in a peptide these contribute to the overall
acid-base properties of the molecule.
Figure 11: Alanylglutamylglycyllysine. This tetrapeptide has one free -amino group, one free -
carboxyl group, and two ionizable R groups. The groups ionized at pH 7.0 are in red.
Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes:
In addition to the peptides formed as products of partial hydrolysis of proteins, many peptides
occur in the free form in living matter. Many of these have intense biologic activity and thus serve
important functions.
No generalizations can be made about the molecular weights of biologically active peptides and
proteins in relation to their functions. Naturally occurring peptides range in length from two to many
thousands of amino acid residues. Even the smallest peptides can have biologically important effects.
Consider the commercially synthesized dipeptide L-aspartyl-L-phenylalanine methyl ester, the
artificial sweetener better known as aspartame or NutraSweet.
Many small peptides exert their effects at very low concentrations. For example, a number of
vertebrate hormones are small peptides. These include oxytocin (nine amino acid residues), which is

15. 14
secreted by the posterior pituitary and stimulates uterine contractions; bradykinin (nine residues),
which inhibits inflammation of tissues; and thyrotropin-releasing factor (three residues), which is
formed in the hypothalamus and stimulates the release of another hormone, thyrotropin, from the
anterior pituitary gland. Some extremely toxic mushroom poisons, such as amanitin, are also small
peptides, as are many antibiotics.
Cys.Tyr.Ile.Gln.Asn.Cys.Pro.Leu.Gly.NH2
Oxytocin
Arg.Pro.Pro.Gly.Phe.Ser.Pro.Phe.Arg
Bradykinin
Pyroglutamyl - histidinyl - prolinamide
Thyrotropin Releasing Factor (TRF)
Glutathione, a typical tripeptide in which the N-terminal glutamate is linked to cysteine via a non-
peptide bond, present in all forms of life, required for action of several enzymes.
(-glutamylcysteinylglycine)
Functions:
 Serve as a component of an amino acid transport system.
 An activator of certain enzymes (acting as Co enzyme for transhydrogenases e.g. glutathione
insulin transhydrogenase).
 Protection of lipids against autoxidation.
 Play important role in detoxification reaction:
e.g. Dichloronitrobenzene + GSH Mercapturic acid (excreted by kidney)
S S

16. 15
Cl
Cl
Cl
S-CH2-CH-COO-
O2NO2N
NH
CO
CH3
GSH
It is most remarkable that these peptides have such potent biological effects, despite the fact that
the amino acids of which they are composed are harmless, non-toxic substances. Clearly it is the
sequence of amino acids in polypeptides that gives them a three dimensional structure or shape and
hence their striking biological effects and specificity.
Slightly larger are small polypeptides and oligopeptides such as the pancreatic hormone insulin,
which contains two polypeptide chains, one having 30 amino acid residues and the other 21.
Glucagon, another pancreatic hormone, has 29 residues; it opposes the action of insulin. Corticotropin
is a 39-residue hormone of the anterior pituitary gland that stimulates the adrenal cortex.
How long are the polypeptide chains in proteins? Human cytochrome c has 104 amino acid
residues linked in a single chain; bovine chymotrypsinogen has 245 residues. At the extreme is titin,
a constituent of vertebrate muscle, which has nearly 27,000 amino acid residues and a molecular
weight of about 3,000,000.
Some proteins consist of a single polypeptide chain, but others, called multisubunit proteins, have
two or more polypeptides associated noncovalently. The individual polypeptide chains in a
multisubunit protein may be identical or different. If at least two are identical the protein is said to be
oligomeric, and the identical units (consisting of one or more polypeptide chains) are referred to as
protomers. Hemoglobin, for example, has four polypeptide subunits: two identical -chains and two
identical -chains, all four held together by noncovalent interactions. Each -subunit is paired in an
identical way with a -subunit within the structure of this multisubunit protein, so that hemoglobin
can be considered either a tetramer of four polypeptide subunits.
A few proteins contain two or more polypeptide chains linked covalently. For example, the two
polypeptide chains of insulin are linked by disulfide bonds. In such cases, the individual polypeptides
are not considered subunits but are commonly referred to simply as chains.
We begin with a description of the fundamental chemical properties of amino acids, peptides, and
proteins.

17. 16
Chemical Reactions of Amino Acids
Amino acids contain amino and carboxyl groups and will thus give reactions characteristic for
these groups. These reactions are widely used for the detection, measurement, and identification of
amino acids.
Chemical Reaction of Amino Acids due to NH2 Group
1) In liver: (Detoxification):
NH2CHC
H
HO
O
COOH
+
CONHCH2COOH
Glycine Benzoic Acid Hippuric Acid
(Toxic) (Non Toxic)
This reaction used for liver function or to excreting toxins from the body.
2) With nitrous acid (HNO2):
This reaction is the basis of the (Van Slyke) method for determination of the amino group in A.A.,
peptides, proteins (proline, hydroxy proline) doesn't react with HNO2.

18. 17
3) Determination of the amino terminal residue of a polypeptide with
fluorodinitrobenzene (Sanger’s reagent):

19. 18
Determination of the amino terminal residue of a polypeptide by the Edman
degradation:
Edman’s regent has an advantage over Sanger’s reagent in that it can be applied repeatedly on the
shortened peptide.
5- With formaldehyde: (Sorensen reaction)
Intermediate in a No. of enzymatic reaction

20. 19
6- Oxidative deamination:
7- With CO2:
Important in the transport of CO2 by blood hemoglobin (Hb)
Chemical Reaction of Amino Acids due to COOH Group
1- Decarboxylation:
Glutamic acid GABA
GABA: -Aminobutryric acid.
This is formed in the brain by decarboxylation of glutamic acid, where it may act as a chemical
mediator in the transmission of the nerve impulse between some neuron.
HN N
CH2CHNH2COOH
HN N
CH2CH2NH2
Histidine Histamine
Histamine: arises from histidine by decarboxylation. It is a vasodilator and is involved in shock
and allergic responses.
2- With NH3 to form amide:
e.g. Asparagine and glutamine.

21. 20
Chemical Reaction of Amino Acids due to (R) Group
1. Ninhydrin reaction:
 Ninhydrin oxidatively decarboxylates α-amino acids to CO2, NH3 and aldehyde with one less
carbon atom than the parent amino acid.
 The reduced ninhydrin then reacts with the liberated ammonia,
forming a blue complex. This reaction is used for visualization of amino acids separated by paper
chromatography.
2. Biuret test:
 This reaction is given by compounds containing two peptide bonds or more.
 A violet colour appears on mixing protein solution with strong NaOH and dilute copper
sulfate.
3. Rosenheim test:
 This test is given by proteins containing indole ring e.g. tryptophan.
4. Xanthoproteic test:
 This test is given by the aromatic amino acids phenylalanine, tyrosine and tryptophan. Yellow
colour is formed when these acids are boiled with concentrated nitric acid. On addition of
alkali, this yellow colour turns orange.
5. Millon’s test:
 This test is given by the amino acid tyrosine. Mixing of tyrosine solution with Millon's reagent
produces a brick red colour. The Millon's reagent consists of nitric acid solution of mercuric
and mercurous nitrites.
6. Sulfur test:
 This test is given by the sulfur-containing amino acids e.g. cysteine and cysteine. A brown or
black precipitate is formed on boiling of these amino acids with strong NaOH and lead acetate
solution. Methionine does not give sulfur test because sulfur is masked by the methyl group.

22. 21
Orders (Levels) of Protein Structure
There Are Several Levels of Protein Structure:
For large macromolecules such as proteins, the tasks of describing and understanding structure
are approached at several levels of complexity, arranged in a kind of conceptual hierarchy. Four levels
of protein structure are commonly defined (Fig. 12). A description of all covalent bonds (mainly
peptide bonds and disulfide bonds) linking amino acid residues in a polypeptide chain is its primary
structure. The most important element of primary structure is the sequence of amino acid residues.
Secondary structure refers to particularly stable arrangements of amino acid residues giving rise to
recurring structural patterns. Tertiary structure describes all aspects of the three-dimensional folding
of a polypeptide. When a protein has two or more polypeptide subunits, their arrangement in space is
referred to as quaternary structure.
Primary structure:
The sequence of amino acids in a protein is called the primary structure of the protein.
Understanding the primary structure of proteins is important because many genetic diseases result in
proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment
of normal function. If the primary structures of the normal and the mutated proteins are known, this
information may be used to diagnose or study the disease.
The exact sequence of amino acids in the polypeptide chain or chains including the exact locations
of the disulfide bridges constitutes the primary structure.
OR, refers to the covalent backbone of the polypeptide chains, including the sequence of
amino acid residues.
Protein structure, from primary to quaternary structure.
Figure 12: Levels of structure in proteins. The primary structure consists of a sequence of amino
acids linked together by peptide bonds and includes any disulfide bonds. The resulting
polypeptide can be coiled into units of secondary structure, such as an -helix. The helix is a part

23. 22
of the tertiary structure of the folded polypeptide, which is itself one of the subunits that make
up the quaternary structure of the multi-subunit protein, in this case hemoglobin.
Secondary structure:
Refers to a regular, recurring arrangement in space of the polypeptide chain along one
dimension.
OR, refers to regularly coiled or zigzag arrangements of polypeptide chains along one dimension.
E.g. Fibrous proteins are typical 2ry structure. The α-helix and the B-pleated sheet are example of
2ry structure.
A- The -helix:
1) The α-helix is a rod like structure with the side chains of the amino acids extending outward from
the central axis of the coiled polypeptide backbone.
2) Hydrogen bonds extend down the spiral from the carbonyl oxygen of one peptide linkage to the
-NH- group of a peptide bond four residues ahead in the primary sequence. All the carbonyl
oxygen's and peptide bonded nitrogen's along the polypeptide backbone are hydrogen bonded in
the
α-helix. These hydrogen bonds are individually weak but collectively are the major forces
stabilizing the helical structure.
3) Each turn of the helix contains 3.6 amino acids; thus amino acid residues spaced three or four
apart in the primary sequence are spatially close together when folded in the α-helix.
4) Proline disrupts the α-helix because its imino group is not geometrically compatible with the
right handed spiral of the α-helix. Large numbers of charged amino acids (e.g. glutamate,
aspartate, histidine, lysine, and arginine) or amino acids with bulky side chains (e.g. valine,
isoleucine and tryptophan) are also incompatible with the α-helix.
5) The α-helical content of proteins can vary widely, ranging from about 75% for myoglobin and
hemoglobin to a virtual absence of helix in chymotrypsin, a digestive enzyme secreted by the
pancreas.

24. 23
B- The -pleated sheet:
1- In some proteins, the polypeptide chains line up side by side to form sheets of molecules, called
the β-pleated sheet.
2- The polypeptide chain is almost fully extended in the β-pleated sheet, rather than being coiled.
The structure is stabilized by H-bonds between different polypeptide chains (interchain bonds), in
contrast to the intrachain H-bonds of the α-helix.
3- Adjacent strands most commonly run in the opposite direction that is an anti-parallel β-sheet. Silk
fibroin is composed almost entirely of this structure. Many globular proteins contain short stretches
of β-pleated sheet in which the polypeptide chain changes direction by folding back on its
α-helix form in an intrachain B bend.
Can be of 2 types:
 Antiparallel-run in an opposite direction of its neighbor (A).
 Parallel-run in the same direction with longer looping sections between them (B).

25. 24
Tertiary structure:
 Refers to how the polypeptide chain is folded in three dimensions, to form the compact, tightly
folded structure of globular proteins. Most globular proteins are tertiary structure. Globular
proteins are spherical in shapes consisting of amount of coils with no regular structure.
Interactions (Forces) Stabilizing Tertiary Structure:
The unique three-dimensional structure of each protein is determined by its amino acid sequence.
Interactions of the amino acid side chain quid the folding of the polypeptide chain to form a compact
structure.
Four types of interactions cooperate in stabilizing the tertiary structures of globular proteins:
1- Hydrophobic interactions:
Amino acids with nonpolar side chains tend to fold into the interior of the protein molecule
where they associate with other hydrophobic amino acids. In contrast, amino acids with polar or
charged side chains tend to be located on the molecules of the solvent (Fig. 13).
2- Hydrogen bonds:
Amino acid side chains containing loosely bound hydrogen's, such as in the alcohol groups of
serine and threonine, can form hydrogen bonds with electron rich atoms such as nitrogen atoms of
histidine or the carbonyl oxygen of carboxyl groups, amide groups, and peptide bonds (Fig. 14).
3- Ionic interactions:
Negatively charged carboxyl groups - COO- can interact with positively charged groups, such
as the -amino (-NH3 +) of lysine (Fig. 14).
4- Covalent cross linkages:
A disulfide bond is a covalent bond between the thiol group (-SH) of each of two cysteine
residues, resulting in the formation of cysteine (Fig. 15). The two cysteines that participate in the
disulfide bond may be separated by many amino acids in the primary sequence of a protein.
However, the folding of the polypeptide chain can bring the cysteine residues in proximity and
allow covalent bonding of their side chains. The disulfide linkage contributes to the stability of
the three dimensional shape of the protein molecule. Many disulfide bonds are found in proteins
that are excreted from the cell. It is thought that these strong, covalent bonds aid in stabilizing the
structure of proteins, preventing them from becoming denatured in the various extracellular
environments.

26. 25
Figure 13: Hydrophobic interactions between amino acids with nonpolar side chains.
Figure 14: interactions of side chains of amino acids through hydrogen bonds and ionic bonds.
Figure 15: Covalent cross linkages.
Quaternary structure:
In proteins containing more than one polypeptide chain, the number as well as the arrangement
of the subunits is called the quaternary structure. The subunits are usually held together by
noncovalent bonds (hydrophobic interactions, hydrogen and ionic bonds). For example, the enzyme
lactate dehydrogenase (LDH) contains four separate polypeptide chains assembled into a tetrmeric
protein.

27. 26
Classification of Proteins
They may be classified on the bases of.......
(1) Overall (3) Solubility
(2) Function. (4) Physical properties.
(1) Overall shape:
Two broad classes of proteins may be distinguished on the basis of their axial ratio (ratio of length to
breadth): In the native state each type of protein molecule has a characteristic three-dimensional
shape, referred to as its conformation.
1- The Fibrous Proteins:
Consist of polypeptide chains arranged in parallel along a single axis, to yield long fibers or sheets.
Fibrous proteins are physically tough and are insoluble in H2O. They are the basic structural elements
in the connective tissue of higher animals. e. g. collagen of tendons and bone matrix, α-keratin of
hair, skin, nails, & elastic of elastic connective tissue.
2- The Globular Proteins:
The polypeptide chains are tightly folded into compact spherical or globular shapes. Most globular
proteins are soluble in aqueous system and usually have a dynamic function in the cell. 2000 different
enzymes, antibodies, some hormones and many proteins having a transport function, e.g. s.alb. & Hb.
are globular proteins.
(2) Proteins carry out diverse functions and may be classified on that basis:
CLASS FUNCTION
Enzymes Proteins with catalytic activity. These are the most varied and the
most highly specialized proteins. Over 2000 different enzymes are
known, each capable of catalyzing a different kind of chemical
reaction e.g. ribonuclease, trypsin.
Transport proteins Carry specific ions or molecules from one organ to another, e.g.
hemoglobin (oxygen), albumin (bilirubin, fatty acids).
Storage proteins Store nutrients or other molecules. e.g. ferritin (iron), casein (milk),
ovalbumin of egg white.
Structural proteins Give biological structure strength or protection. e.g. collagen
(tendon, cartilage and bone), elastin (ligaments) or yellow elastic
tissue.
Contractile proteins Give cells ability to contract, change shape or move about. e.g. actin
(long filamentous protein of globular chain) and myosin, rod-like
shape (muscle).
Defense Defense organisms against invasion by other species or protect them
from injury. e. g. fibrin (blood clotting), antibodies (infection).
Regulatory proteins Regulate cellular or physiological activity. e.g. growth hormone
(growth), histones (gene regulation).
Proteins have unusual
function.
Besides there are other proteins with rather exotic functions which
are not classified. e.g. monellin from an African plant has an
intensely sweet taste, antifreeze protein from an Antarctic fish
prevents blood from freezing. Spiders & silkworms secrete a thick
solution of the protein. Fibroin, which quickly solidifies into an
insoluble thread of exceptional tensile strength used to form webs or
cocoons.

28. 27
(3)- Proteins may also be classified according to Solubility as:
Simple proteins Conjugated or compound Derived proteins.
1- Simple proteins: on hydrolysis gives only amino acids.
A- Albumins:
-These are soluble in water and salt solutions.
-They can be coagulated by heat.
-They are of high biological value, being easily digested and rich in the essential amino
acids.
-The molecular weight of albumin is about 68,000. They are precipitated by full saturation
with ammonium sulfate. Examples include: ovalbumin in egg white, lactalbumin in milk
and serum albumin in blood plasma.
B- Globulins:
-These are insoluble in water, but soluble in salt solutions.
-Globulins can be coagulated by heat and precipitated by half saturation with ammonium
sulfate (NH4)2 SO4.
-They are proteins of high biological value. Their molecular weight is 150,000. Examples
include: many enzymes, ovaglobulins in egg white, lactglobulins in milk and plasma
globulins in blood plasma.
C- Prolamins:
e.g. gladden of egg white, Zein of corn. On hydrolysis give proline + NH3.
- Soluble in 70 - 80% ethanol but insoluble in water and absolute ethanol.
D- Globins:
- formed in nature as globin of hemoglobin, insoluble in H2O but soluble in NH4 OH.
E- Histones:
-Occur as a part of nucleoprotein. Soluble in salt solution but insoluble in NH 4 OH,
coagulated by heat.
F- Albuminoids:
It is a protein of supportive tissue as collagen, elastin and keratin's. Insoluble in water or
neutral solvents.
2- Conjugated proteins: contain chemical components (prosthetic group) a non-
protein particle in addition to amino acids.
A- Nucleoprotein:
- Protein (protamine or histone) + nucleic acid.
-The nucleic acid may be ribonucleic acid (RNA) which is present in ribonucleoprotein
of the ribosomes or deoxyribonucleic acid (DNA) which is present in deoxyribonucleo-
protein of chromatin.
B- Glycoprotein’s:
 These are proteins that have oligosaccharide chains covalently attached to their
polypeptide backbones.
 Glycoproteins occur in most organisms from bacteria to humans. Their carbohydrate
content ranges from 1% to over 85% by weight. The oligosaccharide chains of
glycoproteins perform different functions including the following:
(a) They modulate the physicochemical properties of proteins e.g. solubility, viscosity,
charge and denaturation.
(b) They protect proteins against proteolysis.
(c) They are involved in the biologic activity of the protein e.g. those of human chorionic
gonadotropin (hCG).
(d) They affect insertion of proteins into membranes, intracellular migration and secretion
of proteins.

29. 28
(e) They affect the embryonic development and differentiation.
(f) They may affect sites of metastasis selected by cancer cells.
* Examples of glycoproteins include:
(1) Most plasma proteins except albumins e.g. blood clotting factors.
(2) Some protein hormones e.g. FSH & TSH.
(3) Structural proteins e.g. collagen, laminin and fibronectin.
(4) The blood group substances A and B.
C- Phosphoprotein:
-These are proteins containing a phosphate radical, which is attached to the hydroxyl
group of the amino acid serine.
-Phosphoproteins include caseinogen of milk, vitellins of egg yolk and some enzymes
e.g. phosphorylase and glycogen synthase.
D- Chromoprotein:
 These are proteins containing coloured prosthetic group. They may be
metallochromoproteins (containing metal) or non-metallochromoproteins (containing
no metal).
 Examples of metallochromoproteins include the following:
(a) Iron-containing proteins which are brownish in colour e.g. ferritin & hemosiderin.
(b) Copper- containing proteins which are greenish to bluish in colour e.g. ceruloplasmin.
(c) Metalloporphyrinoproteins, e.g. chlorophyll proteins containing magnesium and hem
proteins containing iron (Hb, myoglobin and cytochromes).
 Examples of non-metallochromoproteins include:
(a) Flavoproteins, containing FMN or FAD, which are yellowish in colour e.g. some
enzymes.
(b) Carotenoid proteins, containing carotenoid prosthetic group e.g. rhodopsin and iodopsin
which are the visual pigments of the retina.
Melanoproteins which are found in the skin, hair and iris giving them brown to black colour.
E- Lipoprotein:
-These are proteins complexes with lipids, in which the protein surrounds the lipids
making them water soluble.
-Examples of lipoproteins include plasma lipoproteins which are:
*Chylomicrons.
* Low density lipoproteins (LDL).
*Very low density lipoproteins (vLDL).
* High density lipoproteins (HDL).
-Plasma lipoproteins are synthesized either in the intestine or the liver and help the transport
of lipids in the blood from one tissue to the other.
F- Metalloprotein:
These are proteins containing metal prosthetic group. Examples include:
a) Ferritin, transferrin and hemosiderin, containing iron.
b) Ceruloplasmin (plasma), erythrocuprin (erythrocytes), hepatocuprin (liver) and cerebrocuprin
(brain), containing copper.
c) The hormone insulin is stored in the form of crystals containing zinc. In addition some enzymes
contain zinc e.g. carbonic anhydrase.
d) Enzymes- containing magnesium, e.g. kinases, phosphatases.
e) Enzymes-containing manganese, e.g. arginase and carboxylases.
1) Derived proteins: formed by the action of heat, enzymes, or partial hydrolysis of simple
and conjugated proteins. Derived proteins include the following:

30. 29
 Primary protein derivatives: These Include proteins resulting from alteration in the structure
of the native proteins without hydrolysis e.g. denatured proteins (metaproteins) and coagulated
proteins.
 Secondary protein derivatives: These include products of hydrolysis of proteins which include
the following:-
(a) Proteoses: These are the first products of hydrolysis of proteins. They have high molecular
weight but smaller than proteins, therefore, they can be precipitated by
concentrated ammonium sulfate.
(b) Peptones: These result from further hydrolysis of proteoses. Peptones are small molecules
which can not be precipitated by concentrated ammonium sulfate.
(c) Small peptides: These are composed of a few amino acids. Examples include: * Dipeptides,
containing 2 amino acids e.g. carnosine & anserine.* Tripeptides, containing 3
amino acids e.g. glutathione.
(d) Amino acids: These are the final products of hydrolysis of proteins.
(4) Proteins are also classified according to physical properties:
Besides, proteins are also classified on the basis of physical properties like electrophoretic
mobility (serum α, B- & -globulin's) or buoyant density (VLDL, HDL, LDL).

31. 30
Fibrous Proteins
Fibrous proteins have structures consisting of several polypeptide chains often tightly associated
one to the other. These proteins are relatively insoluble in most physiologic fluids. Examples of
fibrous proteins are the keratins, Elastin's, and Collagen's.
 The KERATINS:Are the proteins in hair, fingernails, and horny tissues (containing high content
of cystine amino acid). They are the highly insoluble fibrous proteins, and have α-helical
structures. When -keratin is heated, the strong intrachain hydrogen bonds of the α-helix are
broken, and the molecules extend into a parallel pleated sheet. This form is called -keratin.
Although the intrachain H-bonds break, the stable interchain disulfide bonds remain intact. On
cooling, -keratin reforms its intrachain H-bonds and returns to the α-conformation.
 The ELASTIN: It is a yellow fluorescent protein found mostly in ligaments and blood vessel
walls, but is also occurs in small amounts in skin, tendon, and loose connective tissues. Unlike
collagen, fibers of elastin can be stretched to several times their length, and they snap back almost
like a piece of rubber. Tropoelastin is the basic building unit for the elastic fibers. It contains
components having molecular weights ranging from 30000 to 100000. Although elastin and
collagen contain similarly high amounts of glycine and proline and both lack cysteine and
tryptophan, elastin contains less hydroxyproline and no hydroxylysine. The glycine content is
about the same, but the Gly - X - Y repeating unit of collagen is not present. Consequently,
elastin is resistant to hydrolysis by bacterial collagenase.
 The COLLAGENS: Collagen is the most abundant protein in the mammalian body (more than
30%) and also forms the extracellular framework for all multicellular animals. The strong fibers
of collagen form the main structure of bone, teeth, blood vessels, tendons, and cartilage, but
collagen also forms vital parts of the structure of woven sheets like those that occur in skin and
in filtration membranes of the glomerulus. Collagen is important in repair processes such as the
formation of scar tissue in wound healing.
COLLAGENS:
A typical collagen molecule is a long, rigid structure in which three polypeptides (referred to as
"α-chains") are wound around one another in a rope-like triple-helix (Figure). Although these
molecules are found throughout the body, their types and organization are dictated by the structural
role collagen plays in a particular organ. In some tissues, collagen may be dispersed as a gel that gives
support to the structure, as in the extracellular matrix or the vitreous humor of the eye. In other tissues,
collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons. In the
cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen
of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any
direction.
Figure 16: Triple-stranded helix of collagen.
All collagen molecules are organized as triple helices: this structure is also found in two other
proteins, namely acetylcholine esterase and the complement protein C1q. The triple helical structure
of collagen is in Fig.16, each of the three polypeptide chains being designated as -like. Each -
chain twists in a left handed helix with three amino acid residues per turn and the three chains are
wound in a right handed super-helix to form a rod like molecule almost 1.4 nm in diameter.

32. 31
Gly-Leu-Hyp- Gly-Pro-Hyp- Gly-Ala-Hyl-
Figure 17: Amino acid sequence of a portion of the 1-chain of collagen. (Note: Hyp is
hydroxyproline and Hyl is hydroxylysine.).
Types of collagen:
The collagen super-family of proteins includes more than twenty collagen types, as well as
additional proteins that have collagen-like domains. The three polypeptide α-chains are held together
by hydrogen bonds between the chains. Variations in the amino acid sequence of the α-chains result
in structural components that are about the same size (approximately 1000 amino acids long), but
with slightly different properties. These α-chains are combined to form the various types of collagen
found in the tissues. For example, the most common collagen, type I, contains two chains called 1
and one chain called 2 ( 12 2). In this form of collagen, the chains each contain about 1050
aminoacyl residues and the molecule is 300 nm in length, whereas type II collagen contains three
  13). The collagens can be organized into three groups, based on their location and
functions in the body (Table 4).
Table 4: The most abundant types of collagen.
Structure of Collagen:
1) Amino acid sequence: Collagen is rich in proline and glycine, both of which are important in the
formation of the triple-stranded helix. Proline facilitates the formation of the helical
conformation of each α-chain because its ring structure causes "kinks" in the peptide chain.
Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits
into the restricted spaces where the three chains of the helix come together. The glycine residues
are part of a repeating sequence, -Gly-X-Y-, this repeat of glycine is essential to the structure,
because it is the only amino acid small enough to fit in the central core of the molecule. Ends of
the collagen molecule are not, however, in the triple helical form, and they lack glycine. Where
TYPE TISSUE DISTRIBUTION
I
II
II
Fibril-forming:
 Skin, bone, tendon, blood vessels, cornea.
 Cartilage, intervertebral disk, vitreous body.
 Blood vessels, fetal skin.
IV
VII
Network-forming:
 Basement membrane.
 Beneath stratified.
 Squamous epithelia.
IX
XII
Fibril-associated:
 Cartilage.
 Tendon, ligaments, some other tissues

33. 32
X is frequently proline and Y is often hydroxyproline or hydroxylysine (Figure). Thus, most of
the α-chain can be regarded as a polytripeptide whose sequence can be represented as (-Gly-X-
Y-)333.
2) Triple-helical structure: Unlike most globular proteins that are folded into compact structures,
collagen, a fibrous protein, has an elongated, triple-helical structure that places many of its amino
acid side chains on the surface of the triple-helical molecule.[Note: This allows bond formation
between the exposed R-groups of neighboring collagen monomers, resulting in their aggregation
into long fibers.]
3) Hydroxyproline and hydroxylysine: Collagen contains hydroxyl-proline (hyp) and
hydroxylysine (hyl), which are not present in most other proteins. These residues result from the
hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide
chains (Figure). The hydroxylation is, thus, an example of posttranslational modification).
Hydroxyproline is important in stabilizing the triple-helical structure of collagen because it
maximizes interchain hydrogen bond formation. Proline and hydroxyproline together account for
about one third of the X-Y positions so that they form 25% of the total aminoacyl residues. The
high concentration of hydroxyproline is very unusual in proteins. About 90 hydroxyproline
residues are needed per chain to preserve the triple helix at body temperature; if the biosynthesis
of hydroxyproline is impaired, unstable collagen is formed in which the chains tend not to wind
round each other. Most of the hydroxyproline is 4-hydroxyproline, although a small proportion
of 3-hydroxyprolyl residues are also found in the collagen molecules. Type I collagen contains
only one or two residues of 3-hydroxyproline near the carboxyl end of the chain, but other types
of collagen may contain 10% of the total hydroxyproline as the C-3 derivative.
4) Lysyl residues are also hydroxylated and subsequently a glycosyl residue, to which a galactosyl
residue may become attached, is joined in an O-glycosidic linkage. Another, but extra-cellular
reaction undergone by some of the lysyl residues is one of oxidative deamination when the -
amino group is lost and an aldehyde group is formed. This is able to react with free amino groups
in lysyl residues and as these are usually located in other chains, the result being the type of cross-
linking illustrated in Fig.
5) Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be
enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to
the polypeptide chain prior to triple-helix formation.
In summary, whilst the functions of some of the side chain modifications are uncertain, it is now
appreciated that 4-hydroxyproline stabilizes the helix of the molecule and hydroxylysine is essential
for sugar attachment; the carbohydrate chain may be effective in water retention and also in cross-
link formation.

34. 33
Figure 19: Formation of cross-links in collagen.
The role of ascorbic acid in biosynthesis of collagen:
Many researches were done to elucidate the action of ascorbic acid; when guinea-pigs deficient
in ascorbic acid (scorbutic) were studied, it became clear that, they were suffering from an inability
to synthesize collagen efficiently. Furthermore, many of the symptoms of scurvy in man, such as the
weak blood vessels and poor wound healing, appear to be a result of ineffective synthesis of collagen.
It therefore, became apparent that ascorbic acid played a very important role in collagen biosynthesis.
This role has, in recent years, been finally established and ascorbic acid has been shown to be
intimately involved in the formation of hydroxy-prolyl and hydroxy-lysyl residues in collagen. The
chains of the intracellular precursor of collagen, called procollagen, are synthesized on ribosome's
which, as in the case for all secretary proteins, are attached to the rough endoplasmic reticulum. These
chains contain all of the amino acids appropriate to the particular collagen molecule, in the correct
sequence, but there are no hydroxy-prolyl or hydroxy-lysyl residues. Indeed there are no codons in
the genetic code to specify these two amino acids nor are there any tRNA molecules for them. As
the α-chain precursors are formed certain, but not all, of the prolyl and lysyl residues are hydroxylated
in reaction catalyzed by two hydroxylases. Prolyl-hydroxylase has been particularly well studied and
is known to require iron and molecular oxygen as well as ascorbic acid. Another unusual cofactor
for the reaction is 2-oxoglutarate which is oxidized to succinate.

35. 34
Figure 20: Hydroxylation of prolyl residues of pro--chains of collagen by prolylhydroxylase.
It is now believed that the role of ascorbic acid is one of maintaining the iron of prolyl-
hydroxylase in the reduced (Fe)+ form.

36. 35
In all, three enzymes are involved in the process of hydroxylation, forming 4-hydroxyproline, 3-
hydroxyproline or hydroxylysine. The 4-hydroxylase attacks only those prolyl residues which are in
the “Y” position, whereas the 3-hydroxylase hydrox-ylates the prolyl residues in the “X” position, in
the - (Gly- X - Y)n -sequence.
As the lysyl residues are hydroxylated, the glucosyl and galactosyl residues are added to them by
appropriate transferases. This procollagen is assembled in the rough endoplasmic reticulum and
passes through the Golgi apparatus before leaving the cell.
After its release from the cell pro-collagen acts as a substrate for three enzymes (see Fig.), two
peptidases which catalyze the hydrolytic removal of the non helical regions at the N- and C- terminal
ends of the -chains and lysyl oxidase, a copper containing oxygenase, that catalyses the conversion
of some lysyl residues into the corresponding aldehyde, lysinal.

37. 36
Elastin:
In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is
a connective tissue protein with rubber-like properties. Elastic fibers composed of elastin and
glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments.
They can be stretched to several times their normal length, but recoil to their original shape when the
stretching force is relaxed.
A. Structure of elastin
Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin, which is a
linear polypeptide, composed of about 700 amino acids that are primarily small and nonpolar (for
example, glycine, alanine, and valine). Elastin is also rich in proline and lysine, but contains only a
little hydroxyproline and no hydroxylysine. Tropoelastin is secreted by the cell into the extracellular
space. There it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a
scaffold onto which tropoelastin is deposited. [Note: Mutations in the fibrillin gene are responsible
for Marfan's syndrome.] Some of the lysyl side chains of the tropoelastin polypeptides are
oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains
plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine
cross-link (Figure). This produces elastin an extensively interconnected, rubbery network that can
stretch and bend in any direction when stressed, giving connective tissue elasticity.

38. 37
Globular Proteins
Globular Hemeproteins:
Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic
group. The role of the heme group in each protein is dictated by the environment created by the three
dimensional structure of the protein. For example, the heme group of the cytochrome functions as an
electron carrier that is alternately oxidized and reduced. In contrast, the heme group of the enzyme
catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide.
In hemoglobin and myoglobin, the two most abundant heme-proteins in humans, the heme group
serves to reversibly bind oxygen.
A- Structure of Heme:
1- Heme is a complex of protoporphyrin IX and ferrous iron (Fe+2). The iron is held in the center
of the heme molecule by bonds to the four nitrogen’s of the porphyrin ring.
2- The Fe+2 of heme can form two additional bonds, one on each side of the planar porphyrin ring.
For example, in cytochrome C these fifth and sixth coordination positions are occupied by a
histidine and methionine group of the protein. In myoglobin and hemoglobin, one position is
coordinated to a histidine of the protein, whereas the other position is available to bind oxygen.
3- Oxidation of the heme component of myoglobin and hemoglobin to the ferric (Fe+3) state forms
metmyoglobin and methemoglobin. Neither of these oxidized proteins can bind oxygen, but
instead they contain water at the sixth coordinate position of Fe+3.
4-Heme is a cyclic tetrapyrrole which consists of 4 pyrrole rings linked by 4-alpha-methylene
bridges (=CH-).
5-The pyrrole rings are given the numerals 1, 11, 111 and IV, while
the outer carbons of the pyrrole rings are given the numerals 1 through 8. The substituents
replacing the hydrogen atoms at the 8 positions
determine whether a tetrapyrrole is heme or a related compound. In
heme these are methyl (M), vinyl (V) and propionate (P) groups
arranged in the order M, V, M, V, M, P, P, M.

39. 38
B- Structure and Function of Myoglobin:
1- Myoglobin, a hem protein located primarily in heart and skeletal muscle, functions both as a
reservoir for oxygen and as an oxygen carrier that increases the rate of transport of oxygen
within the muscle cell.
2- Myoglobin comprises a single polypeptide chain that is structurally similar to the individual
polypeptide chains of the hemoglobin molecule. This homology allows myoglobin to serve as
a simple model for interpreting some of the more complex properties of Hb.
3- Myoglobin is a compact molecule with approximately 75% of its polypeptide chain folded into
eight stretches of α-helix. These α-helical regions are labeled A to G in FIG. Four of this
segment is terminated by the presence of proline, whose five-member ring cannot be
accommodated in a α-helix. The other regions of α-helix are interrupted by bends and loops
stabilized by hydrogen and ionic bonds.
4- The interior portion of the myoglobin molecule is composed almost entirely of nonpolar amino
acids. For example, the side chains of alanine, valine, leucine, isoleucine, methionine, and
phenylalanine are packed closely together in the center of the molecule, forming a structure
stabilized by hydrophobic interactions among these clustered residues. In contrast charged
amino acids are located almost exclusively on the surface of the myoglobin, where they can
form hydrogen bonds with water.
5- The heme group of myoglobin sits in a crevice in the protein. This cavity is lined with nonpolar
amino acids, except for two histidine residues. One of the histidines, termed the proximal
histidine, binds directly to the iron of heme. The second, or distal histidine, does not directly
interact with the heme groups but helps to stabilize the ferrous form of the iron porphyrin. The
protein, or globin, portion of myoglobin thus creates a special microenvironment for the heme
that allows the reversible binding of oxygen without the simultaneous oxidation of the ferrous
iron.

40. 39
C- Structure and Function of Hemoglobin:
1- Hemoglobin is found exclusively in the red blood cells, where its main function is to transport
oxygen from the lungs to the capillaries of the tissues.
2- Hemoglobin A, the major hemoglobin in adults, comprises four polypeptide chains (two α-
chains and two β-chains, α 2 β 2) held together by non-covalent interactions. Each sub-unit has
a helical structure and heme-binding pocket similar to that described for myoglobin. However,
the tetrameric hemoglobin molecule is structurally and functionally more complex than
myoglobin. For example, Hb can transport CO2 from the tissues to the lung and carry O2 from
the lungs to the cells of the body. Further, the binding of allosteric effectors regulates the
oxygen binding properties of hemoglobin.
Figure 21: A. Structure of hemoglobin showing the polypeptide backbone.
B. Simplified drawing showing the helices.
3- The hemoglobin tetramer can be envisioned as comprising two identical dimers, α 1 β1 and α 2
β2 (where the numbers refer to dimer 1 and dimer 2). The two polypeptide chains within each
dimer are held tightly together. In contrast, the two dimers are able to move with respect to
each other. The interactions between these mobile dimers are different in deoxyhemoglobin
compared to oxyhemoglobin, and the two dimers occupy different relative positions in
deoxyhemoglobin and oxyhemoglobin:

41. 40
Saturation with oxygen (percent)
A) The deoxy-form of hemoglobin is called the T- or taut form. The α 1β1 and α2 β 2 dimers
interact through a network of ionic bonds and hydrogen bonds that constrain the movement
of the polypeptide chains.
B) The binding of oxygen to hemoglobin causes rupture of some of the ionic bonds and
hydrogen bonds between the α 1 β 1 and α 2 β 2 dimers.
This leads to a structure called the R or relaxed form in which the polypeptide chains have more
freedom of movement.
D- Binding of Oxygen to Hemoglobin:
Oxygen saturation curve for myoglobin and hemoglobin show important differences:
A) Myoglobin has a higher oxygen affinity than hemoglobin. The partial pressure of oxygen needed
to achieve half saturation of the binding site (P50) is approximately 1 mm Hg for myoglobin and
26 mm Hg for hemoglobin. The more tightly oxygen binds, the lower is the P50.
B) The oxygen dissociation curve for myoglobin has a hyperbolic shape. This reflects the fact that
myoglobin reversibly binds a single molecule of oxygen. Thus oxygenated (MbO2) and
deoxygenated (Mb) myoglobin exist in a simple equilibrium:
Mb + O2 MbO2
The equilibrium is shifted to the right or the left as oxygen is added to or removed from the
system.
Figure 22: Oxygen dissociation curves for myoglobin and hemoglobin.
C) The oxygen dissociation curve for hemoglobin is sigmoidal in shape, indicating that the sub-units
cooperate in binding oxygen. This cooperative binding results from the fact that the binding of
an oxygen molecule at one heme increases the oxygen affinity of the remaining heme groups in
the same hemoglobin molecule. Although it is difficult for the first oxygen molecule to bind to
hemoglobin, subsequent binding of oxygen occurs with a high affinity as shown by the steep
upward curve in the region near 20 to 30 mm Hg.
Saturationwith
oxygen(percent)

42. 41
Figure 23: Hemoglobin binds oxygen with increasing affinity.
Figure 24: Transport of oxygen and CO2 by hemoglobin.

43. 42
1. Effect of 2, 3-bisphosphoglycerate on oxygen affinity: 2, 3-Bisphosphoglycerate (2, 3-BPG)
is an important regulator of the binding of oxygen to hemoglobin. It is the most abundant organic
phosphate in the red blood cell, where its concentration is approximately that of hemoglobin. 2,3-
BPG is synthesized from an intermediate of the glycolytic pathway.
A- Binding of 2, 3-BPG to deoxyhemoglobin: 2, 3-BPG decreases the oxygen affinity of
hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding
stabilizes the taut conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be
represented schematically as:
B- Binding site of 2,3-BPG: One molecule of 2,3-BPG binds to a pocket, formed by the two β-
globin chains, in the center of the deoxyhemoglobin tetramer (Figure 25). This pocket contains
several positively charged amino acids that form ionic bonds with the negatively charged
phosphate groups of 2, 3-BPG. [Note: A mutation of one of these residues can result in
hemoglobin variants with abnormally high oxygen affinity.] 2,3-BPG is expelled on oxygenation
of the hemoglobin.
Figure 25: Synthesis of 2, 3-bisphophoglycerate.
E- Minor Hemoglobin’s:
1- Hemoglobin A1C:
Under physiologic conditions, glucose reacts nonenzymically with the N-terminal amino
groups of the B-chain of HbA to form hemoglobin A1C (Fig. 26). Hemoglobin A1C constitutes an
average of about 5% of the total hemoglobin of the erythrocyte. However, in individuals with
diabetes mellitus the amount is elevated twofold to threefold. The rate of formation of HbA1C is
proportional to the concentration of glucose in the blood. The glycosylation of Hb is not reversible.
Therefore, once formed, Hb1C persists for the life span of the erythrocyte. Thus, the total HbA1C
in a population of red blood cells reflects the average glucose concentration during the previous 6
to 10 weeks. The levels of HbA1C can be used as an index of long-term control of hyperglycemia
during the treatment of diabetes.

44. 43
2- Hemoglobin A2 (HbA2):
HbA2 is a minor component of normal adult hemoglobin, first appearing about 12 weeks after
birth and constituting about 2.5% of the total hemoglobin. It comprises two α-chains and two -
chains.
3- Fetal hemoglobin (HbF):
HbF is a tetramer with two α-chains identical to those in HbA, plus two -chains. The -chains
is similar in amino acid sequence to β-chains of HbA but differ in 37 amino acids.
Fig. 26: Hemoglobin A1C.
ao) HbF is the major hemoglobin found in the fetus and newborn. During the last months of fetal
life, HbF accounts for about 60% of the total hemoglobin in the erythrocyte. HbA synthesis
starts at about the eight month of pregnancy and gradually replaces HbF.
bo) Under physiologic conditions fetal hemoglobin (HbF) has a higher affinity for oxygen than does
HbA owing to HbF’s binding only weakly to 2, 3-DPG. Since 2, 3-DPG serves to reduce the
affinity of hemoglobin for oxygen, this weak interaction between 2, 3 DPG and HbF results in a
higher oxygen affinity for HbF relative to HbA. In contrast, if both hemoglobin's are stripped of
there 2, 3-DPG, HbA and HbF have a similar affinity for oxygen.
co) The high oxygen affinity facilitates the transfer of oxygen from the maternal circulation across the
placenta to the red blood cells of the fetus. Fig. shows the relative production of each type of
hemoglobin chain during fetal and postnatal life.

45. 44
Hemoglobinopathies:
Hemoglobinopathies have traditionally been defined as a family of disorders caused either by
production of a structurally abnormal hemoglobin molecule or by synthesis of insufficient quantities
of normal hemoglobin. Sickle cell anemia and the thalassemia syndromes are two representative
hemoglobinopathies that can have severe clinical consequences. Sickle cell anemia results from
production of hemoglobin with an altered amino acid sequence, whereas the thalassemias are caused
by decreased production of normal hemoglobin. It is now known that some mutations lead both to
alteration of globin structure and to decrease synthesis. These latter mutations are relatively rare and
will not be discussed.
A. Sickle cell disease (hemoglobin S disease)
1- Sickle cell anemia, also called sickle cell disease, is the most common disorder resulting from the
production of variant hemoglobin. It primarily occurs in the black population, affecting 1 in 500
newborn infants in the United States. Sickle cell anemia is a homozygous recessive disorder
occurring in individuals who have inherited two mutant genes (one from each parent) that code
for synthesis of the B-chains of the globin molecules.
2- Heterozygous, representing one of ten American blacks, have one normal and one sickle cell gene.
The blood cells of such heterozygous contain both HbS and HbA. These individuals have sickle
cell trait; they usually do not show clinical symptoms.
3- A molecule of HbS contains two normal α-chains and two mutant β-chains in which glutamate at
position six has been replaced with valine.

46. 45
4- During electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive
electrode) than does HbA. This altered mobility of HbS is due to the absence of two negatively
charged glutamate residues in the B-chains, thus rendering HbS less negative than HbA.
Electrophoresis of hemoglobin obtained from lysed red blood cells is routinely used in the
diagnosis of sickle cell trait & sickle cell disease.
5- The substitution of the non-polar side chain of valine for a charged glutamate residue results in a
pronounced decrease in the solubility of HbS in its deoxygenated form. The molecules aggregate
to form fibers that deform the red cells into a crescent or sickle shape. Such sickle cells frequently
block the flow of blood in the small diameter capillaries. This interruption in the supply of oxygen
leads to localized anoxia (oxygen deprivation), which causes pain and eventually death of cells in
the vicinity.

47. 46
B- Thalassemias:
1- The thalassemias are a group of hereditary hemolytic diseases in which there is an imbalance in
the synthesis of globin chains. Normally, synthesis of the α- and β- chains is coordinated so that
each-chain has a B-chain partner. This leads to the formation of α 2B2 globin tetramers of HbA.
In the thalassemias, the synthesis of either the α- or β- chain is defective. For example, α -
thalassemia syndromes are a group of defects in which the synthesis of α-chains is decreased or
absent. The synthesis of the unaffected β-chain continues, however, resulting in the accumulation
of B4 tetramers (HbH) that tend to precipitate.
2- In the more sever B-thalassemia disorders; synthesis of β-chains is decreased, whereas α-chain
synthesis is normal. This leads to the precipitation of aggregates of α-chains, which causes the
premature death of cells destined to become mature red blood cells.
3- The decreased synthesis of globin chains seen in various -and -thalassemias is not the result of
a single type of gene mutation. Rather, each of these syndromes may be caused by a variety of
mutations that have the common feature of interrupting the normal process of protein synthesis.
-thalassemias are most often due to gene deletions, whereas -thalassemias are frequently caused
by nucleotide substitutions or deletion of one or several nucleotides.
4- Individuals who are homozygous for gene mutations that produce
β-thalassemia are designated o or thalassemia major. These patients are severely anemic and
require regular transfusions of blood. Although this treatment is lifesaving, the cumulative effect
of the transfusions is iron overload (a syndrome known as hemosiderosis), which typically causes
death between the ages of 15-25 years.
5- Individuals who are heterozygous for β-thalassemia are termed B+ or thalassemia minor. These
individuals make some B-chains and usually do not require specific treatment.

48. 47
Our understanding of protein structure and function has been derived from the study of many
individual proteins. To study a protein in detail, the researcher must be able to separate it from other
proteins and must have the techniques to determine its properties. The necessary methods come from
protein chemistry, a discipline as old as biochemistry itself and one that retains a central position in
biochemical research.
Proteins can be separated and purified:
A pure preparation is essential before a protein’s properties and activities can be determined.
Given that cells contain thousands of different kinds of proteins, how can one protein are purified?
Methods for separating proteins take advantage of properties that vary from one protein to the next,
including size, charge, and binding properties.
The source of a protein is generally tissue or microbial cells. The first step in any protein
purification procedure is to break open these cells, releasing their proteins into a solution called a
crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or
to isolate specific organelles (Fig. 27).

49. 48
Figure 27: Subcellular fractionation of tissue. A tissue such as liver is first mechanically
homogenized to break cells and disperse their contents in an aqueous buffer. The sucrose
medium has an osmotic pressure similar to that in organelles, thus preventing diffusion of water
into the organelles, which would swell and burst.
(a) The large and small particles in the suspension can be separated by centrifugation at different
speeds, or
(b) Particles of different density can be separated by isopycnic centrifugation. In isopycnic
centrifugation, a centrifuge tube is filled with a solution, the density of which increases from
top to bottom; a solute such as sucrose is dissolved at different concentrations to produce the
density gradient. When a mixture of organelles is layered on top of the density gradient and the
tube is centrifuged at high speed, individual organelles sediment until their buoyant density
exactly matches that in the gradient. Each layer can be collected separately.
Once the extract or organelle preparation is ready, various methods are available for purifying
one or more of the proteins it contains. Commonly, the extract is subjected to treatments that separate
the proteins into different fractions based on a property such as size or charge, a process referred to
as fractionation. Early fractionation steps in purification utilize differences in protein solubility,
which is a complex function of pH, temperature, salt concentration, and other factors. The solubility
of proteins is generally lowered at high salt concentrations, an effect called “salting out.” The addition
of a salt in the right amount can selectively precipitate some proteins, while others remain in solution.
Ammonium sulfate ((NH4)2SO4) is often used for this purpose because of its high solubility in water.
A solution containing the protein of interest often must be further altered before subsequent
purification steps are possible. For example, dialysis is a procedure that separates proteins from
solvents by taking advantage of the proteins’ larger size. The partially purified extract is placed in a
bag or tube made of a semipermeable membrane. When this is suspended in a much larger volume of
buffered solution of appropriate ionic strength, the membrane allows the exchange of salt and buffer
but not proteins. Thus dialysis retains large proteins within the membranous bag or tube while
allowing the concentration of other solutes in the protein preparation to change until they come into
equilibrium with the solution outside the membrane. Dialysis might be used, for example, to remove
ammonium sulfate from the protein preparation.
The most powerful methods for fractionating proteins make use of column chromatography,
which takes advantage of differences in protein charge, size, binding affinity, and other properties
(Fig. 28). A porous solid material with appropriate chemical properties (the stationary phase) is held

50. 49
in a column, and a buffered solution (the mobile phase) percolates through it. The protein-containing
solution, layered on the top of the column, percolates through the solid matrix as an ever-expanding
band within the larger mobile phase (Fig. 28). Individual proteins migrate faster or more slowly
through the column depending on their properties.
Figure 28: Column chromatography.
For example, in cation-exchange chromatography (Fig. 29a), the solid matrix has negatively
charged groups. In the mobile phase, proteins with a net positive charge migrate through the matrix
more slowly than those with a net negative charge, because the migration of the former is retarded
more by interaction with the stationary phase. The two types of protein can separate into two distinct
bands. The expansion of the protein band in the mobile phase (the protein solution) is caused both by
separation of proteins with different properties and by diffusional spreading. As the length of the
column increases, the resolution of two types of protein with different net charges generally improves.
However, the rate at which the protein solution can flow through the column usually decreases with
column length. And as the length of time spent on the column increases, the resolution can decline as
a result of diffusional spreading within each protein band.
Size exclusion chromatography separates proteins according to size. In this method, large
proteins emerge from the column sooner than small ones—a somewhat counterintuitive result. The
solid phase consists of beads with engineered pores or cavities of a particular size. Large proteins
cannot enter the cavities, and so take a short (and rapid) path through the column, around the beads.
Small proteins enter the cavities, and migrate through the column more slowly as a result (Fig. 29b).
Affinity chromatography is based on the binding affinity of a protein. The beads in the column
have a covalently attached chemical group. A protein with affinity for this particular chemical group
will bind to the beads in the column, and its migration will be retarded as a result (Fig. 29c).
A modern refinement in chromatographic methods is HPLC, or high-performance liquid
chromatography. HPLC makes use of high-pressure pumps that speed the movement of the protein
molecules down the column, as well as higher-quality chromatographic materials that can withstand
the crushing force of the pressurized flow. By reducing the transit time on the column, HPLC can
limit diffusional spreading of protein bands and thus greatly improve resolution.

51. 50
Figure 29: Three chromatographic methods used in protein purification.
(a) Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric
charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound
charged groups; those with bound anionic groups are called cation exchangers, and those with
bound cationic groups are called anion exchangers. Ion-exchange chromatography on a cation
exchanger is shown here. The affinity of each protein for the charged groups on the column is
affected by the pH (which determines the ionization state of the molecule) and the concentration
of competing free salt ions in the surrounding solution. Separation can be optimized by gradually
changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient.
(b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size.
The column matrix is a cross-linked polymer with pores of selected size. Larger proteins migrate
faster than smaller ones, because they are too large to enter the pores in the beads and hence take
a more direct route through the column. The smaller proteins enter the pores and are slowed by
their more labyrinthine path through the column.
(c) Affinity chromatography separates proteins by their binding specificities. The proteins retained
on the column are those that bind specifically to a ligands cross-linked to the beads. (In
biochemistry, the term “ligand” is used to refer to a group or molecule that binds to a

52. 51
macromolecule such as a protein.) After proteins that do not bind to the ligand are washed
through the column, the bound protein of particular interest is eluted (washed out of the column)
by a solution containing free ligand.
Proteins can be Separated and Characterized by Electrophoresis:
Another important technique for the separation of proteins is based on the migration of charged
proteins in an electric field, a process called electrophoresis. These procedures are not generally used
to purify proteins in large amounts, because simpler alternatives are usually available and
electrophoretic methods often adversely affect the structure and thus the function of proteins.
Electrophoresis is, however, especially useful as an analytical method.
Gel electrophoresis
The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be
by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The
nature of the separation depends on the treatment of the sample and the nature of the gel.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers
loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis)
maintains polypeptides in a denatured state once they have been treated with strong reducing agents
to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and
SH]) and thus allows separation of proteins by their molecular weight. Sampled proteins become
covered in the negatively charged SDS and move to the positively charged electrode through the
acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are
thus separated according to size (usually measured in kilodaltons, kDa). The concentration of
acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better
the resolution of lower molecular weight proteins. The lower the acrylamide concentration was the
better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along
the gel for most blots.
Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a
commercially available mixture of proteins having defined molecular weights, typically stained so as
to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at
different speeds. These different rates of advancement (different electrophoretic mobilities) separate
into bands within each lane.
The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately
in proportion to their charge-to-mass ratio. The migration of a protein in a gel during electrophoresis
is therefore a function of its size and its shape.
SDS binds to most proteins in amounts roughly proportional to the molecular weight of the
protein, about one molecule of SDS for every two amino acid residues. The bound SDS contributes
a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring
on each protein a similar charge-to-mass ratio. In addition, the native conformation of a protein is
altered when SDS is bound, and most proteins assume a similar shape. Electrophoresis in the presence
of SDS therefore separates proteins almost exclusively on the basis of mass (molecular weight), with
smaller polypeptides migrating more rapidly. After electrophoresis, the proteins are visualized by

53. 52
adding a dye such as Coomassie blue, which binds to proteins but not to the gel itself (Fig. 16). Thus,
a researcher can monitor the progress of a protein purification procedure as the number of protein
bands visible on the gel decreases after each new fractionation step. When compared with the
positions to which proteins of known molecular weight migrate in the gel, the position of an
unidentified protein can provide an excellent measure of its molecular weight (Fig. 30). If the protein
has two or more different subunits, the subunits will generally be separated by the SDS treatment and
a separate band will appear for each.
It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single
sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they
have neutral net charge) in the first dimension, and according to their molecular weight in the second
dimension.
Figure 30: Electrophoresis: (a) Different samples are loaded in wells or depressions at the top of the
polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel
minimizes convection currents caused by small temperature gradients, as well as protein
movements other than those induced by the electric field. (b) Proteins can be visualized after
electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins
but not to the gel itself. Each band on the gel represents a different protein (or protein subunit);
smaller proteins move through the gel more rapidly than larger proteins and therefore are found
nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA polymerase
from E. coli. The first lane shows the proteins present in the crude cellular extract. Successive
lanes (left to right) show the proteins present after each purification step. The purified protein
contains four subunits, as seen in the last lane on the right.

54. 53
Figure 31: Estimating the molecular weight of a protein.

55. 54
It is apparent from the preceding description that the primary, secondary, and tertiary structures
are the basis for the chemical, physical, and biological properties of proteins. When normal
conformation is altered but not accompanied by a rupture of peptide bonds, the protein is denatured
and its distinctive properties and biological activity also may be changed.
Denaturation can take many forms; the tertiary structure may be unfolded, the secondary structure
may assume varying degrees of random coil, or disulfide bonds may be cleaved. The most sensitive
index of denaturation is a loss of biological activity. Changes in solubility occur with more extreme
changes, the coagulation of egg white being obvious examples.
Factors that denature proteins are temperature (50-60 oC), extremes in pH (below 4 or above 10),
high conc. of organic solvents such as alcohol's, acetone, or ether, and urea or guanidine HCl.
Denaturation may also result from exposure to UV light as well as from mechanical agitation, heavy
metals, and detergents.
Denaturation May be either Reversible or Irreversible:
Obviously, coagulated egg white is an extreme example and represents an irreversible change.
Denaturing agents can disrupt the H-, ionic, or hydrophobic bonds that stabilize the structure of
protein molecules. Therefore, denaturation often leads to an irreversible destruction of all quaternary,
tertiary, and secondary structures, producing disordered polypeptide chains.
Reversal of denaturation is called renaturation and was first extensively studied using
ribonuclease.
a) Ribonuclease, an enzyme that hydrolyzes RNA, comprises a single polypeptide chain that
contains four disulfide bonds. In the presence of denaturing agents, such as urea or guanidine
HCl, theses cystine (-S-S) residues can be reduced with β-mercaptoethanol to cysteine (-SH)
residues. The polypeptide of the denatured ribonuclease assumes a random structure that has no
enzymatic activity.
b) Removal of urea and β-mercaptoethanol in the presence of air leads to oxidation of the
sulfhydryls of the denatured enzyme and spontaneous refolding into the native structure. This
shows that the information necessary to specify the unique tertiary structure of ribonuclease is
contained in the amino acid sequence of the enzyme.

56. 55
Structural Aspects of Specific Proteins
Plasma Proteins:
Normal plasma contains 60 to 80 g / L (6-8 g / dl) of protein. Of this,
3-5 g. /dl are albumin and 1.5 - 3.0 g. / dl is composed of a mixture of globulins. The plasma proteins
usually are classified on the basis of solubility and electrophoretic separation into the six categories
listed below:
TYPES AND FUNCTIONS % SPECIAL PROPERTIES
Albumin 55
Transport of organic anions, osmotic
pressure, bind Ca+2.
α1-globulin's 5 Glycoproteins, HDL.
α 2-globulin's 9
Haptoglobin (Hb, ceruloplasmin (Cu+2 -
transport), VLDL.
β-globulin's 13 Transferrin (Fe+2 transport), LDL.
Fibrinogen 7 Blood clotting.
γ-Globulin's 11 Immunoglobulins.
Most clinical electrophoretic analyses of serum are carried out with cellulose acetate paper as a
support and a barbital buffer of pH 8.6.
ALBUMIN:
This protein has an isoelectric point of 4.8 and thus has a considerable -ve charge at a physiologic
pH, which explains the relatively high anodal mobility of albumin on the plasma protein
electrophoretogam. Albumin is a globular protein consisting of a single polypeptide chain, and it has
a molecular weight of about 66300.
The two main functions of albumin are to transport small molecules through the plasma and
extra-cellular fluid and to provide osmotic pressure within the capillary. Many metabolites, such as
free fatty acids and bilirubin, are poorly soluble in water. Yet they must be shuttled through the blood
from one organ to another so that they can be metabolized or excreted. A carrier is required to
enhance the solubility of these substances in plasma so that they can be transported through this
aqueous medium. Albumin fulfills this function and serves as a nonspecific transport protein.
Moreover, albumin binds those poorly soluble drugs such as aspirin, digitalis, the cumarin
anticoagulants, and barbiturates so that they also are efficiently carried through the blood stream.
Also albumin binds small anions and cations. Indeed, about 50% of the calcium in the plasma
exists as a complex with albumin. A second vital function of albumin is that it provides 80% of the
osmotic pressure effect of the plasma proteins. Osmotic pressure is the main force that draws
interstitial fluid back into the capillary at its venous end.
-GLOBULINS:
There are two classes of α-globulin in plasma, known as α1 and α2. The main α1 - globulin is
glycoproteins and HDL. The main α2-globulin is haptoglobin, the transport protein for any
hemoglobin that escape into the plasma; ceruloplasmin, the copper blood coagulation; glycoproteins,
and lipoproteins of VLDL.
GLOBULIN:
The major B-globulins are transferrin (the iron transport protein) and LDL.
-GLOBULIN:
The -globulin fraction is made up of immunoglobulins (IgG, IgA, IgM, IgD, and IgE).

57. 56
Biological Value of Proteins:
A- The biological value of a dietary protein is a measure of its ability to provide the essential amino
acids required for tissue maintenance. Proteins from animal sources (meat, poultry, milk, fish)
have a high biological value because they contain all the essential amino acids in the proportions
similar to those required for synthesis of human tissue proteins.
B- Proteins from plant sources (wheat, corn, rice, beans) typically have a lower biological value than
animal proteins. However, proteins from different plant sources may be combined in such a way
that the result is equivalent in nutritional value to animal protein. for example, corn (lysine
deficient but methionine rich) may be combined with kidney beans (methionine poor but lysine
rich) to produce a complete protein of high biological value. Thus, eating foods with different
limiting amino acids at the same meal results in a dietary combination with a higher biological
value than either of the component proteins. The limiting amino acid is defined as the essential
amino acid present in the lowest amount compared with the optimal amount required.
Nitrogen Balance:
Nitrogen balance occurs when the amount of nitrogen consumed equals the nitrogen excreted in
the urine, sweat, and feces. Most healthy adults are normally in nitrogen balance.
A- Positive nitrogen balance:
Occurs when nitrogen intake exceeds nitrogen excretion. It is observed in situations in which
tissue growth occurs, for example, in children, pregnancy, or during recovery from an emaciating
illness.
B- Negative nitrogen balance:
Occurs when nitrogen losses are greater than nitrogen intake. It is associated with inadequate
dietary protein, lack of an essential amino acid, stresses such as trauma, burns, illness, or surgery.

58. 57
Metabolic Defects in Amino Acid Metabolism
Inborn errors of metabolism are commonly caused by mutant genes that generally result in
abnormal proteins, most often enzymes. The inherited defects may be expressed as a total loss of
enzyme activity or, more frequently, as a partial deficiency in catalytic activity. Without treatment,
the inherited defects of amino acid metabolism almost invariably result in mental retardation or other
developmental abnormalities as a result of harmful accumulation of metabolites. Although more than
50 of these disorders have been described, many are rare, occurring in less than 1 per 250,000 in most
populations (Figure 34). Collectively, however, they constitute a very significant portion of pediatric
genetic diseases (Figure 35). Phenylketonuria is the most important disease of amino acid metabolism
because it is relatively common and responds to dietary treatment.
Figure 34: Incidence of inherited diseases of amino acid metabolism. [Note: Cystinuria is the most
common genetic error of amino acid transport.]

59. 58
Figure 35: Summary of the metabolism of amino acids in humans. Genetically determined enzyme
deficiencies are summarized in white boxes. Nitrogen-containing compounds derived from
amino acids are shown in small, yellow boxes. Classification of amino acids is color coded:
Red = glucogenic; brown = glucogenic and ketogenic; green = ketogenic. Compounds in Blue
All Caps are the seven metabolites to which all amino acid metabolisms converge.
A. Phenylketonuria:
Phenylketonuria (PKU), caused by a deficiency of phenylalanine hydroxylase (Figure 36), PKU
is the most common clinically encountered inborn error of amino acid metabolism (prevalence

60. 59
1:15,000). Biochemically, it is characterized by accumulation of phenylalanine (and a deficiency of
tyrosine). Hyperphenylalaninemia may also be caused by deficiencies in any of the several enzymes
required to synthesize BH4, or in dihydropteridine (BH2) reductase, which regenerates BH4 from BH2
(Figure 37). Such deficiencies indirectly raise phenylalanine concentrations, because phenylalanine
hydroxylase requires BH4 as a coenzyme. BH4 is also required for tyrosine hydroxylase and
tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such
as serotonin and catecholamines. Simply restricting dietary phenylalanine does not reverse the central
nervous system (CNS) effects due to deficiencies in neurotransmitters. Replacement therapy with
BH4 or L-DOPA and 5-hydroxytryptophan (products of the affected tyrosine hydroxylase– and
tryptophan hydroxylase–catalyzed reactions) improves the clinical outcome in these variant forms of
hyperphenylalaninemia, although the response is unpredictable.
Figure 36: A deficiency in phenylalanine hydroxylase results in the disease phenylketonuria (PKU).
 Screening of newborns for a number of the amino acid disorders using a few drops of blood is
possible; however, exactlywhich disorders are screened for currently varies from stateto state,
and only phenylketonuria screening is mandated by all states.

61. 60
Figure 37: Biosynthetic reactions involving amino acids and tetrahydrobiopterin.
1. Characteristics of classic PKU:
a) Elevated phenylalanine: Phenylalanine is present in elevated concentrations in tissues,
plasma, and urine. Phenyllactate, phenylacetate, and phenylpyruvate, which are not normally
produced in significant amounts in the presence of functional phenylalanine hydroxylase,
are also elevated in PKU (Figure 37). These metabolites give urine a characteristic musty
(“mousey”) odor. [Note: The disease acquired its name from the presence of a phenylketone
(now known to be phenylpyruvate) in the urine.]
b) CNS symptoms: Mental retardation, failure to walk or talk, seizures, hyperactivity, tremor,
microcephaly, and failure to grow are characteristic findings in PKU. The patient with
untreated PKU typically shows symptoms of mental retardation by the age of one year, and
rarely achieves an IQ greater than 50 (Figure 25). [Note: These clinical manifestations are
now rarely seen as a result of neonatal screening programs.]
c) Hypopigmentation: Patients with phenylketonuria often show a deficiency of pigmentation
(fair hair, light skin color, and blue eyes). The hydroxylation of tyrosine by tyrosinase, which
is the first step in the formation of the pigment melanin, is competitively inhibited by the
high levels of phenylalanine present in PKU.

62. 61
Figure 37: Pathways of phenylalanine metabolism in normal individuals and in patients with
phenylketonuria.
Figure 38: Typical intellectual ability in untreated PKU patients of different ages.

63. 62
2. Neonatal screening and diagnosis of PKU: Early diagnosis of phenylketonuria is important
because the disease is treatable by dietary means. Because of the lack of neonatal symptoms,
laboratory testing for elevated blood levels of phenylalanine is mandatory for detection.
However, the infant with PKU frequently has normal blood levels of phenylalanine at birth
because the mother clears increased blood phenylalanine in her affected fetus through the
placenta. Normal levels of phenylalanine may persist until the newborn is exposed to 24 to 48
hours of protein feeding. Thus, screening tests are typically done after this time to avoid false
negatives. For newborns with a positive screening test, diagnosis is confirmed through
quantitative determination of phenylalanine levels.
3. Antenatal diagnosis of PKU: Classic PKU is a family of diseases caused by any of 400 or more
different mutations in the gene that codes for phenylalanine hydroxylase (PAH). The frequency
of any given mutation varies among populations, and the disease is often doubly heterozygous,
that is, the PAH gene has a different mutation in each allele. Despite this complexity, prenatal
diagnosis is possible.
4. Treatment of PKU: Most natural protein contains phenylalanine, and it is impossible to satisfy
the body's protein requirement when ingesting a normal diet without exceeding the phenylalanine
limit. Therefore, in PKU, blood phenylalanine is maintained close to the normal range by feeding
synthetic amino acid preparations low in phenylalanine, supplemented with some natural foods
(such as fruits, vegetables, and certain cereals) selected for their low phenylalanine content. The
amount is adjusted according to the tolerance of the individual as measured by blood
phenylalanine levels. The earlier treatment is started, the more completely neurologic damage
can be prevented. [Note: Treatment must begin during the first seven to ten days of life to prevent
mental retardation.] Because phenylalanine is an essential amino acid, overzealous treatment that
results in blood phenylalanine levels below normal should be avoided because this can lead to
poor growth and neurologic symptoms. In patients with PKU, tyrosine cannot be synthesized
from phenylalanine and, therefore, it becomes an essential amino acid that must be supplied in
the diet. Discontinuance of the phenylalanine-restricted diet before eight years of age is
associated with poor performance on IQ tests. Adult PKU patients show deterioration of IQ
scores after discontinuation of the diet (Figure 39). Lifelong restriction of dietary phenylalanine
is, therefore, recommended. [Note: Individuals with PKU are advised to avoid aspartame, an
artificial sweetener that contains phenylalanine.]
5. Maternal PKU: When women with PKU who are not on a low- phenylalanine diet become
pregnant, the offspring are affected with “maternal PKU syndrome.” High blood phenylalanine
levels in the mother cause microcephaly, mental retardation, and congenital heart abnormalities
in the fetus. Some of these developmental responses to high phenylalanine occur during the first
months of pregnancy. Thus, dietary control of blood phenylalanine must begin prior to
conception, and must be maintained throughout the pregnancy.

64. 63
Figure 39: Changes in IQ scores after discontinuation of low-phenylalanine diet in patients with
phenylketonuria.
B. Maple syrup urine disease:
Maple syrup urine disease (MSUD) is a rare (1:185,000), autosomal recessive disorder in which
there is a partial or complete deficiency in branched-chain α-keto acid dehydrogenase, an enzyme
complex that decarboxylates leucine, isoleucine, and valine. These amino acids and their
corresponding α-keto acids accumulate in the blood, causing a toxic effect that interferes with brain
functions. The disease is characterized by feeding problems, vomiting, dehydration, severe metabolic
acidosis, and a characteristic maple syrup odor to the urine. If untreated, the disease leads to mental
retardation, physical disabilities, and even death.
1) Classification: The term “maple syrup urine disease” includes a classic type and several variant
forms of the disorder. The classic from is the most common type of MSUD. Leukocytes or cultured
skin fibroblasts from these patients show little or no branched-chain α-keto acid dehydrogenase
activity. Infants with classic MSUD show symptoms within the first several days of life. If not
diagnosed and treated, classic MSUD is lethal in the first weeks of life. Patients with intermediate
forms have a higher level of enzyme activity (approximately 3–15% of normal). The symptoms
are milder and show an onset from infancy to adulthood. Patients with the rare thiamine-dependent
variant of MSUD achieve increased activity of branched-chain α-keto acid dehydrogenase if given
large doses of this vitamin.
2) Screening and diagnosis: As with PKU, antenatal diagnosis and neonatal screening are available,
and most affected individuals are compound heterozygotes.
3) Treatment: The disease is treated with a synthetic formula that contains limited amounts of
leucine, isoleucine, and valine, sufficient to provide the branched-chain amino acids necessary for
normal growth and development without producing toxic levels. Early diagnosis and lifelong
dietary treatment is essential if the child with MSUD is to develop normally. [Note: Branched-
chain amino acids are an important energy source in times of metabolic need, and individuals with
MSUD are at risk of decompensation during periods of increased protein catabolism.]
C. Albinism:
Albinism refers to a group of conditions in which a defect in tyrosine metabolism results in a
deficiency in the production of melanin. These defects result in the partial or full absence of pigment
from the skin, hair, and eyes. Albinism appears in different forms, and it may be inherited by one of
several modes: autosomal recessive (primary mode), autosomal dominant, or X-linked. Complete
albinism (also called tyrosinase-negative oculocutaneous albinism) results from a deficiency of
tyrosinase activity, causing a total absence of pigment from the hair, eyes, and skin. It is the most

65. 64
severe form of the condition. In addition to hypopigmentation, affected individuals have vision
defects and photophobia (sunlight hurts their eyes). They are at increased risk for skin cancer.
D. Homocystinuria
The homocystinurias are a group of disorders involving defects in the metabolism of
homocysteine. The diseases are inherited as autosomal recessive illnesses, characterized by high
plasma and urinary levels of homocysteine and methionine and low levels of cysteine. The most
common cause of homocystinuria is a defect in the enzyme cystathionine β-synthase, which converts
homocysteine to cystathionine (Figure 40). Individuals who are homozygous for cystathionine β-
synthase deficiency were exhibit ectopia lentis (displacement of the lens of the eye), skeletal
abnormalities, premature arterial disease, osteoporosis, and mental retardation. Patients can be
responsive or nonresponsive to oral administration of pyridoxine (vitamin B6), a coenzyme of
cystathionine β-synthase. Vitamin B6–responsive patients usually have a milder and later onset of
clinical symptoms compared with B6-nonresponsive patients. Treatment includes restriction of
methionine intake and supplementation with vitamins B6, B12, and folate.
Figure 40: Enzyme deficiency in homocystinuria.

66. 65
E. Alkaptonuria
Alkaptonuria is a rare metabolic disease involving a deficiency in homogentisic acid oxidase,
resulting in the accumulation of homogentisic acid. [Note: This reaction occurs in the degradative
pathway of tyrosine.] The illness has three characteristic symptoms: homogentisic aciduria (the
patient's urine contains elevated levels of homogentisic acid, which is oxidized to a dark pigment on
standing, Figure A), large joint arthritis, and black ochronotic pigmentation of cartilage and
collagenous tissue (Figure B). Patients with Alkaptonuria are usually asymptomatic until about age
40. Dark staining of the diapers sometimes can indicate the disease in infants, but usually no
symptoms are present until later in life. Diets low in protein, especially in phenylalanine and tyrosine,
help reduce the levels of homogentisic acid, and decrease the amount of pigment deposited in body
tissues. Although Alkaptonuria is not life-threatening, the associated arthritis may be severely
crippling.
Further readings:
 Lippincott (Review Illustrated Biochemistry).
 Lehninger's (Principles of Biochemistry).
 Harper's (Illustrated Biochemistry).
 Basic Concepts in Biochemistry.