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PHARM 281 INTRODUCTORY BIOCHEMISTRY & MOLECULAR BIOLOGY I
PROTEINS
Dr Charles Ansah
Room C119 New Pharmacy Block
Department of Pharmacology
Faculty of Pharmacy & Pharmaceutical Sciences
College of Health Sciences
KNUST
PROTEINS
CLASSIFICATION OF PROTEINS
•May be classified as follows:
•Composition
•Simple (contain only amino acids
•Conjugated (contain additional substances)
•Molecular weight
•Low (5000-20,000)
•Medium (20,000-50,000)
•High (50,000-several millions)
•Molecular shape
•Fibrous (long in proportion to their diameter)
•Globular (less asymmetric)
•Function
•Enzymes
•Structural
•Antibodies
•Source
•Tissue protein
•Plant
•Bacteria
•Viral
•Physicochemical properties
•Solubility
soluble
insoluble
•Thermal stability
stable
unstable
Generally however, proteins are classified as:
•SIMPLE
•Albumins
Water soluble, and soluble in dilute salt solutions
Precipitated with full saturation in ammonium sulphate
Eg. Plasma albumin
•Globulins
Soluble in dilute salt solutions
Insoluble in water and strong salt solutions
•Scleroproteins
Insoluble in aqueous solutions eg. Keratin, collagen, fibrin
•Protamines
Contain High proportion of arginine
Of low MW, not coagulated by heat, soluble in water to give
appreciably alkali solution
•Histones
•Soluble in water to give weakly alkali solution, conjugated
as nucleoprotein
•CONJUGATED PROTEINS:
•Nucleoproteins-nucleic acids
eg chromosomes
•Glycoproteins or mucoproteins
carbohydrate derivatives-blood group substances
•Lipoproteins-plasma lipoproteins, components of cell
Membranes and subcellular organells
•Phosphoproteins-phosphoesters with serine or
threonine residues eg. casein
•Flavoproteins-flavine-adenine dinucleotide, various
reduction and oxidative enzymes
•Haemoproteins-Iron-porphyrin (haem) groups eg. Hb,
Myoglobin, cytochrome c
•Metalloproteins-containing metal groups eg carbonic
anhydrase
•Proteins
•Greek – proteios – of 1° importance
•Polymers of amino acids linked by peptide
bonds.
•Proteins are the most important of all
biological compounds.
•Components of Proteins
•A copolymer is a polymer made from more
than one type of monomer molecule.
•Twenty different a-amino acids can link to
form polypeptides.
•Distribution of Body Proteins
20%
20%
10%
10%
50%
50%
20%
20%
Muscle
Muscle
Bone
Bone
Skin
Skin
Other
Other
•Protein for Energy
•Prefer to use fat, CHO for energy
•CHO and fat are protein sparing
•EXCEPTIONS
–During prolonged strenuous exercise,
about 15% of the muscles need met with
protein (break down own tissue)
–If protein intake is inadequate, body
protein  energy e.g. starvation
•Blood Levels
•Total plasma proteins – 6.0-8.4 g/dL
–Albumin – 3.5-5.0 g/dL
–Globulin – 2.3-3.5 g/dL
•Proteins-Properties & Functions
•Size
•Proteins are extremely large natural polymers
with molecular weights reaching several
million.
•Compare a typical organic molecule -benzoic
acid (C6H5COOH MW = 132).
•The small protein haemoglobin has the
formula C2952H4664O832N812S8Fe4.
•Its molecular weight = 65,000.
•Size contd.
•Proteins are too large to pass through cell
membranes and remain trapped in the cells
where they are made.
•In disease or trauma, cells are damaged
and proteins can escape.
•Detection of proteins in urine indicates
kidney damage. Heart attack releases
specific heart cell proteins into the blood.
•Size of Some Important Proteins
Protein





Insulin
Cytochrome c
Hemoglobin
Gamma globulin
Myosin
Molecular wt
6,000
16,000
65,000
176,000
800,000
No. of aa residues
51
104
574
1320
6100
•Properties contd.
•Proteins are linear polymers built of monomer
units called amino acids
•Proteins contain a wide range of functional
groups.
•Proteins can interact with one another and with
other biological macromolecules to form complex
assemblies
•Some proteins are quite rigid, whereas others
display limited flexibility
•Linear Polymer
•Function of a protein is directly
dependent on its three-dimensional
structure
•Proteins spontaneously fold up into
three-dimensional structures that are
determined by the sequence of amino
acids in the protein polymer.
•Functional Groups
•alcohols, thiols, thioethers, carboxylic
acids, carboxamides, and a variety of
basic groups.
•combined in various sequences, this
array of functional groups accounts for
the broad spectrum of protein function.
•Interaction + macromolecules
•assemblies include
–macro-molecular machines that carry out
the accurate replication of DNA,
– the transmission of signals within cells,
and
–many other essential processes.
•Rigidity & Flexibility
•Rigid units can function as structural
elements in the cytoskeleton (the internal
scaffolding within cells) or in connective
tissue.
•may act as hinges, springs, and levers
that are crucial to protein function,
•The assembly of proteins with one
another and with other molecules into
complex units,
•and to the transmission of information
within and between cells
•Other properties of proteins
•Sedimentation
-a protein containing solution centrifuged
at sufficiently high speed will have its molecules
settled at a constant rate when the centrifugal
force exceeds the dispersant forces on the molecules
•pH
-The pH determines the properties of the protein such
as solubility, viscousity and enzymatic activity.
•Immunofluorescent histochemistry
-the precise location of an antigenic substance can be determined
by an antibody that reacts specifically with it.
•Electrophoresis
-At any pH other than the IEP, a protein will migrate in an electric
field. The differential rates of migration can be used to separate
proteins.
•Hydrolysis
Hydrolysis (enzymatic or heat) of the
amides regenerates the amino acids:
H
O
N
C
R
R
N
C
H
O
H
O
N
C
R
R
H
N
C
H
O
N
•The amide linkage is split as indicated.
•Regeneration of component amino acids
The very large protein is broken down into
smaller, water soluble components:
O
H2N
COH
H2N
R
O
R
H2N
COH
O
COH
H2N
R
R
COH
O
•These small molecules may move through the
organism to be reassembled elsewhere.
•General functions of proteins
•Most versatile macromolecules in living
systems
•serve crucial functions in essentially all
biological processes
–catalysts,
–transport and store other molecules such as
oxygen,
– provide mechanical support and immune
protection,
–generate movement,
– transmit nerve impulses, and
– control growth and differentiation
•Functional Roles of Proteins
•Dynamic Functions
Transport, metabolic control, contraction,
and catalysis of chemical transformations.
•Structural Functions
provide the matrix for bone and connective
tissue
give structure and form to the human
organism.
•Dynamic Functions I
•Enzymatic Catalysis
•Enzymes-dynamic proteins. almost all biological
reactions are enzyme catalyzed . Allows the
reaction to occur at a rate compatible with life.
•Transport
•Haemoglobin and myoglobin
•transport oxygen in blood and in muscle
respectively
•Transferrin
•transports iron in blood.
•Albumin
•many drugs and xenobiotics compounds are
transported bound to albumin.
•Others transport hormones in blood from their site of
synthesis to their site of action
•Dynamic Functions II
Protective
Role
–Immunoglobulins and interferons
act against bacterial or viral infection.
–Fibrin
formed where required to stop the loss of
blood on injury to the vascular system.
Metabolic Control
–Many hormones are proteins.
–Protein hormones include insulin, thyrotropin,
somatotropin (growth hormone), luteinizing
hormone, and follicle stimulating hormone.
Important peptide hormones include
adrenocorticotropin, antidiuretic hormone,
glucagon, and calcitonin.
•Dynamic Functions III
•Contractile Mechanisms
–Myosin and actin
function in muscle contraction.
•Control And Regulation Of Gene Transcription And
Translation
–histone proteins closely associated with DNA, the
repressor and enhancer proteins that control gene
expression, and the proteins that form a part of the
ribosomes.
•Structural Functions
•brick-and-mortar" roles
–collagen and elastin,
form
the matrix for bone, ligaments, connective
tissue and skin
Provide structural strength and elasticity to
organs
a-keratin –
Keratin
is the 1º component of human hair, nails,
skin, and tooth enamel – fibrous sulfur-containing
protein.
Protein Structure
•Levels of protein structure
Primary structure
–The amino acid sequence in a polypeptide chain
Secondary
structure
–Consists of local regions of polypeptide chains
formed into structures that are usually stabilized
by hydrogen bonds
Tertiary structure
–Involves folding of the secondary elements into
an overall three-dimensional conformation
Quaternary structure
–Combination of 2 or more subunits each
composed of a polypeptide chain
Protein
Organization
Four
levels of
organization
a- helix
–Primary structure
–Secondary
structure
Myoglobin
–Tertiary structure
–Quaternary
structure
Hemoglobin
•Primary Structure
1˚
structure = specification of the sequence
of amino acids i.e. the order in which amino acid
residues are linked together in a protein.
Note:
since every polypeptide begins with free
amino group, this is called the N-terminus. The
opposite end of the polypeptide has a free carboxyl
group, called the C-terminus.
N and C terminal of polypeptides
R
H
H
N
H
C
C
H
O
Amino
or N
terminus
N
R
R
H
C
H
C
O
N
C
C
N
R
O
C
C
OH
H
O
H
Carboxyl
or C
terminus
•Amino Acid Sequences Have Direction
•Leu-enkephalin - an opioid peptide, modulates the perception of pain.
• reverse pentapeptide, Leu-Phe-Gly-Gly-Tyr (LFGGY), is a different molecule and
shows no such effects
Polypeptide chains
consists
of a regularly repeating part,
called the main chain or backbone
and a variable part, comprising the
distinctive side chains
•Why know the sequence of amino acids
in a polypeptide chain?
• Elucidating its mechanism of action (e.g., the
catalytic mechanism of an enzyme)
– proteins with novel properties can be
generated by varying the sequence of
known proteins.
• Second, amino acid sequences determine the
three-dimensional structures of proteins.
– sequence is the link between the genetic
message in DNA and the three-dimensional
structure that performs a protein's biological
function.
•Oxytocin & Vasopressin
•ADH and oxytocin each have nine (9) amino acids.
•Each has cysteine residues at amino acid positions 1 and 6.
•These cysteine residues form a disulfide bond with one another to
create a cyclic six amino acid ring with 3 amino acid residues hanging
off.
•ADH and oxytocin share 7 amino acids in common and differ only at
amino acid positions 3 and 8.
•Oxytoxin is Isoleucine-3, Leucine-8 while ADH is Phenylalanine-3,
Arginine-8.
•Functions of Oxytocin & ADH
Oxytocin
stimulates contraction of uterine smooth
muscle. It is secreted during labor to effect
delivery of the fetus.
Oxytocin also stimulates contraction of smooth
muscle in the mammary glands (myoepithelial
cells).
ADH
in low doses controls the reabsorption of
water by the distal tubules of the kidneys and
regulates the osmotic content of blood.
At high doses, ADH causes contraction of
arterioles and capillaries, especially those of the
coronary vessels, to produce localized increases in
blood pressure
Receptors, V1 – blood vessels, V2- kidney
10 structure of Insulins Used in the Treatment of DM
Species
A8
A9
A10
B30
Human
Thr
Ser
Ile
Thr
Cow
Ala
Ser
Val
Ala
Pig
Thr
Ser
Ile
Ala
Sheep
Ala
Gly
Val
Ala
Horse
Thr
Gly
Ile
Ala
Dog
Thr
Ser
Ile
Ala
Chicken*
His
Asn
Thr
Ala
Duck*
Glu
Asn
Pro
Thr
*Positions 1 and 2 of B chain are both Ala in chicken and duck; whereas
in the other species in the table, position 1 is Phe and position 2 is Val in
B chain.
•Insulin lispro
In
insulin lispro, reversal of the proline at B-28 and
the lysine at B-29 results in more rapid dissolution of
this insulin to a dimer and then to a monomer that is
absorbed more rapidly after subcutaneous injection
•Pharmacokinetics
•Secondary Structure
Polypeptides
fold in a series of stages. The first
level of folding is called the secondary (2˚)
structure.
One of the most common 2˚ folding patterns is
called the alpha-helix , discovered by Pauling and
Corey.
–Alpha helix: Hydrogen bonds can form readily
between C=O groups in the backbone and N-H
groups four amino acid residues further along
the chain.
–This regular pairing pulls the polypeptide into
a helical shape that resembles a coiled ribbon.
20 structure contd
•Another common folding pattern is called
beta pleated sheet .
•Some protein regions remain in random
coil, no regular pattern of secondary
structure.
•Different proteins have different degrees
of alpha helix, beta sheet, and random
coil .
•Silk is a protein stabilized entirely by
pleated sheet; keratin (in hair) is
stabilized entirely by alpha helix. Most
proteins have some of both.
Alpha helix
•Hydrogen-Bonding Scheme For an a helix
the
CO group of residue n forms a hydrogen
bond with the NH group of residue n+ 4.
Structure of an α-helix
The
polypeptide
backbone is folded into a
spiral that is held in
place by hydrogen bonds
(black dots) between
backbone oxygen atoms
and hydrogen atoms.
 Note that all the
hydrogen bonds have
the same polarity. The
outer surface of the
helix is covered by the
side-chain R groups.
Beta sheet
•A simple two-stranded b sheet with antiparallel b strands.
• A sheet is stabilized by hydrogen bonds (black dots)
between the b strands.
•The planarity of the peptide bond forces a b sheet to be
pleated; hence, this structure is also called a b pleated
sheet, or simply a pleated sheet.
Side
view of a b sheet showing how the
R groups protrude above and below the
plane of the sheet.
Fibrous Proteins
Highly elongated protein molecules whose shapes
are dominated by a single type of secondary
structure.
Example
Characteristics
1. Coiled Coil
Keratin
durable, insoluble, unreactive
2. b Sheet
Silk
soft, flexible
3. Triple Helix
Collagen
strong, high tensile strength
Type
Keratin
• principal component of hair, nails, wool, horns,
hooves, scales, feathers, shells
• a keratin - in mammals
• b keratin - in birds and reptiles
The a-keratin chain is an a-helix. Pairs of a-helix chains are
interwound to form a two-chain coiled coil. The chains wind in a
left-handed sense.
Each a-keratin chain consists of ~310 residues having a 7-residue
repeat:
a-b-c-d-e-f-g
where residues a and d are nonpolar
Silk - a b sheet
• consists of
antiparallel b sheets
• 6-residue repeat
(-Gly-Ser-Gly-Ala-Gly-Ala-)n
• The b sheets stack
to form a
microcrystalline array.
Collagen - a triple helix
• Single collagen molecule
contains 3 polypeptide chains.
• Each chain is a left-handed
helix (3 residues/turn).
• 3 helical chains are twisted
together in a right-handed
manner to form a superhelical
structure.
• Many varieties - eg., Type I has
two a1 and one a2 chains
Collagen - distinctive amino acid
composition
30% Gly and 15-30% Pro or Hyp (hydroxyproline)
(-Gly-X-Pro-) repeats or (-Gly-X-Hyp-) repeats
Hyp (4-hydroxyproline)
Pro
O
O
C
N
CH
H2C
CH2
C
H2
C
prolyl
hydroxylase
(requires
ascorbic acid)
CH
N
H2C
CH2
C
HO
H
Collagen Diseases
• Scurvy (vitamin C deficiency) - improper fibers,
skin lesions, fragile blood vessels, poor wound
healing, due to decreased Hyp formation
• Osteogenesis imperfecta (brittle bone disease)
(OI) a group of heritable disorders with an incidence
of about 1 in 10, 000- abnormal bone formation in
infants, varies from mild to lethal.
• Defect due to mutation in the genes for procollagen
Type I, single base change in the codon for glycine
resulting in the disruption of the triple helical
structure.
• Ehler-Danlos syndrome - hyperextensibility of joints
and skin (“loose” skin), mutations: Gly replaced with
Ser or Cys
Schematic Views of a-Helices
• A ball-and-stick model.
• A ribbon depiction.
• A cylindrical depiction.
Ferritin
• Ferritin, an iron-storage protein, is built
from a bundle of a helices.
Major Histocompatibility Complex
• Model of binding site in class I MHC (major
histocompatibility complex) molecules, which are involved in
graft rejection.
• A sheet comprising eight antiparallel b strands (green)
forms the bottom of the binding cleft, which is lined by a pair
of a helices (blue).
• A disulfide bond is shown as two connected yellow spheres.
The MHC binding cleft is large enough to bind a peptide 8
10 residues long.
Tertiary Structure
Polypeptides
continue folding beyond the
formation of secondary structure.
It
is only with the complete, compact folding
into tertiary (3°) structure that they attain
their "native conformation" and become active
proteins (as a result of the creation of active
sites).
Forces
that contribute to tertiary folding
include:
–hydrogen bonds
–hydrophobic bonds
–ionic bonds
–sulfhydryl bonds (-S-S- bonds). These are
especially important, because they are
covalent bonds and quite strong compared to
H-bonds.
Tertiary Structure
Protein Folding
Protein
synthesis generates a linear
sequence that has to be folded with
hydrophilic groups on the outside and
hydrophobic groups buried (if it is water
soluble).
The
primary structure determines the
folding pattern.
Given
the number of possible structures
it is not possible that the protein tests
every one of them to find the lowest
energy state.
Protein Folding
It
is thought that secondary structures, called
‘molten globules’, facilitate the folding process.
Another
problem is that as proteins are
synthesised hydrophobic regions must not be
exposed to an aqueous environment or they
will associate to form aggregates.
This
is achieved by chaperones that bind to
hydrophobic regions and subsequently detach
to allow correct folding.
Protein Folding contd
This
process allows the correct folding of even
large proteins since these fold sequentially as
they are synthesised.
Some
proteins require chaperonins that
enclose the protein to be folded in a cavity
away from the rest of the cell.
Chaperones
and chaperonins do not direct
protein folding but simply provide conditions
where it can occur properly.
In
cells exposed to a near lethal temperature
rise heat shock proteins are synthesised.
These allow existing proteins to refold correctly.
Examples include Hsp 70 and Hsp 60
Prion diseases and protein folding
Novel
pathogens composed entirely of proteins
A number of neurological degenerative diseases are known to
be caused by prions
–These include Creutzfeldt–Jacob disease (CJD) and kuru
in humans and scrapie (Bovine spongiform
encephalopathy,BSE) in sheep.
–Mad cow disease is also caused by a prion.
Although
they are infectious no nucleic acid has been
identified and it is now thought that a protein infectious agent
or prion is responsible.
In
scrapie there is a normal brain protein (PrPc) which
becomes converted to the scrapie form (PrPsc).
These
have the same primary structure but different
secondary and tertiary structures.
Prion Diseases
It
is suggested that the prion form converts the
normal form to the prion form, i.e. the process is
autocatalytic.
There
are two possible mechanisms for this
–The association of the normal form with the prion form
may be sufficient to cause the change
–There may be an involvement of a chaperone and ATP
in the unfolding and refolding
Mutations
in the normal gene for PrP may make
the formation of PrPsc more likely.
Assignment !
Alzheimer’s Disease ?
Pathophysiology?
Which
protein?
Are there any herbals available for
the management of Alzhemer’s?
Domains
A
long protein sequence frequently folds into a
series of compact, semi-independent regions called
domains.
Each
domain has a hydrophobic core and a
hydrophilic exterior and generally are 100-150
amino acids in length.
Domains
by a
of a single protein are usually connected
stretch
of polypeptide chain lacking a usual
secondary structure (random coil) or
a cleft or less dense region of tertiary structure.
Sometimes a binding site is found in a cleft between
domains.

Domains contd
Domains
are frequently associated with a
specific function of the protein.
For
example: binding sites for two different
substrates or a substrate and effector could be in
two different domains.
Example:
Glyceraldehyde-3-phosphate
dehydrogenase..one domain binds NAD+ and the
second domain binds glyceraldehyde-3phosphate.
The cell-surface protein, CD4
cluster of differentiation
•Cell surface protein found on some cells of the
immune system.
•Has an extracellular and cytoplasmic portions.
•(HIV) attaches itself to the extracellular portion,
which comprises of four similar domains of
approximately 100 amino acids each
Quaternary Structure
Some
proteins are made of multiple
polypeptide subunits, which must be
assembled together after each individual
polypeptide has reached its 3° structure.
Examples:
–Hemoglobin (blood protein involved in oxygen
transport) has four subunits .
–Pyruvate dehydrogenase (mitochondrial
protein involved in energy metabolism) has 72
subunits.
Immunoglobulins (Igs)
Consist
of 2 heavy and 2 light chains.
 A disulfide bond joins a L chain to a H
chain and the two L-H chain pairs are
bound together by two disulfide bonds
between the H chains.
The variable regions of an L and H chain
come together to form the antigen binding
site of the immunoglobulin.
Structure of Antibodies
Structure of antibodies
The
heavy and light chains come together to
form Fab domains, which have the antigenbinding sites at the ends.
The
two heavy chains form the Fc domain.
The Fab domains are linked to the Fc domain
by flexible linkers
Myoglobin and Hemoglobin
Both
proteins are involved in oxygen transport.
myoglobin
= intracellular protein in muscle
hemoglobin
= intracellular protein in red blood cells
Why study them?
vital
proteins in human health
valuable
model in studying protein structure, binding, function
Myoglobin
153
a.a. residues
MW
16,700
X-ray
structure, 1959
eight
a-helices
contains
a heme group
–iron atom
–porphyrin ring system
Heme group
Fe(II)
coordinated to
N atoms in porphyrin
ring
Fe(II) binds O2
–with O2 = scarlet
–no O2 = dark purple
Fe(II)
can be oxidized
to Fe(III) - dark
brown, does not bind
O2
Myoglobin Function
Major
physiological role is to facilitate oxygen
transport in muscle.
Essentially
solutions.
increases oxygen solubility in aqueous
In
aquatic mammals, myoglobin also functions to
store oxygen (10-fold more in seals and whales)
Reversible
binding of O2 to myoglobin (Mb)
Mb + O2
MbO2
Hemoglobin
intracellular
protein in red blood cells
physiological
binds
tissues
function is to transport oxygen
oxygen in lungs and releases oxygen into
quaternary
structure
–tetrameric protein
–two a-subunits and two b subunits - a2b2
–each subunit contains a heme group
–Fe(II) binds O2
with
no
O2 = scarlet
O2 = dark purple
Haemoglobinopathies
•Over 300 variations of amino acid sequences of normal
adult haemoglobin (HbA) have been reported.
•Differ by:
-insertion of incorrect amino acid into either b or a-chain
during protein synthesis
•Haemoglobin variants may function normally or abnormally
depending on the nature and position of the substitution
Haemoglobin variants
Name
Hammersmith
Bristol
Bibba
Savannah
Philly
Mutation
Phe CD1(42)b  Ser
Val E11(67) b  Asp
Leu H19(136) a  Pro
Gly B6(24) b  Val
Tyr C1(35) a  Phe
Boston
Milwaukee
Iwate
Yakima
His E7(58) a  Tyr
Val E11(67) b  Glu
His F8(87) a  Tyr
Asp G1(99) b  His
Kansas
Asn G4(102) b  Thr
Sickle-cell anemia
Glu A6(6) b  Val
(hemoglobin S)
Effect
Weakens heme binding
Weakens heme binding
Disrupts the H helix
Disrupts the B-E helix interface
Disrupts hydrogen bonding at the a1-b1
interface
Promotes methemoglobin formation
Promotes methemoglobin formation
Promotes methemoglobin formation
Disrupts a hydrogen bond that stabilizes
the T conformation
Disrupts a hydrogen bond that stabilizes
the R conformation
Deoxyhemoglobin S forms insoluble
filaments that deform erythrocytes.
Mutant Val on one b subunit interacts
in hydrophobic pocket of another b
subunit , forming linear polymers.
Haemoglobin variants
Sickle Cell Disease
Most
common hereditary blood disorder
Most
common of the conditions is sickle cell anaemia (SCA)
affecting mainly the black population.
In
SCA, the Haemoglobin called HbS contains normal a-
chains but its b-chain contain valine instead of
glutamate at residue 6, ie, a hydrophobic amino acid
replaces an acidic one.
The
hydrophobic valine is able to interact with the b85-Phe
and b88-leu of an adjacent deoxy HbS.
Consequences of the alteration:
•Modification of the Hb conformation, stacking of 280
million Hb molecules within each erythrocyte altered
by the production of fibrous aggregates.
•Change
in shape of erythrocytes from a biconcave
disc to a crescent or sickle shape on deoxygenation
In homozygotes the erythrocytes interact to form
clumps, occlusion of capilaries and consequent
reduction in blood flow. Organ damage!
•
SCA
is characterized by episodes of pain, chronic
hemolytic anemia and severe infections, usually
beginning in early childhood
Sickle cell anaemia
Under
certain
conditions such as
low O2 levels, RBCs
with HbS distort into
sickle cells
The
sickled cells can
block small vessels
producing
microvascular
occlusions which may
cause necrosis of the
tissue
Sickle Cell Anaemia
Detection
–gel electrophoresis. Because sickle
hemoglobin lacks a glutamate, it is less
acidic than HbA. Hemoglobin HbS,
therefore, does not migrate as rapidly
towards the anode as does HbA.
–It is also possible to diagnose sickle-cell
anemia by recombinant DNA techniques.
SCA – Management
-
a combination of fluids, analgesics,
antibiotics and transfusions are used
to treat symptoms and
complications.
–
–Hydroxyurea, an antitumor drug, has
been shown to be effective in
preventing painful crises.
–Hydroxyurea induces the formation of
fetal Hb (HbF) - a Hb normally found in
the fetus or newborn - which, when
present in individuals with SCA,
prevents sickling.
Degradation of Proteins
Cells
have both extracellular and intracellular pathways
for degrading proteins.
The major extracellular pathway is the system of
digestive proteases, which break down ingested proteins
to polypeptides in the intestinal tract.
 endoproteases such as trypsin and chymotrypsin,
which cleave the protein backbone adjacent to basic and
aromatic residues
exopeptidases, which sequentially remove residues
from the N-terminus (aminopeptidases) or C-terminus
(carboxypeptidases) of proteins; and
 peptidases, which split oligopeptides into di- and
tripeptides and individual amino acids.
These small molecules are then transported across the
intestinal lining into the bloodstream
Protein
Degradation: intracellular
Pathways
The
life span of intracellular proteins varies
from as short as a few minutes for mitotic
cyclins, which help regulate passage through
mitosis, to as long as the age of an organism for
proteins in the lens of the eye.
Cells
have several intracellular proteolytic
pathways for degrading:
misfolded or denatured proteins,
 normal proteins whose concentration must be
decreased,
 and foreign proteins taken up by the cell.
One
major intracellular pathway involves
degradation by enzymes within lysosomes,
membrane-limited organelles whose interior is
acidic.
Protein
Degradation
Distinct
from the lysosomal pathway are
cytosolic mechanisms for degrading
proteins.
The best-understood pathway, the
ubiquitin-mediated pathway, involves two
steps: •addition
of a chain of ubiquitin molecules
to an internal lysine side chain of a target
protein
•proteolysis
of the ubiquitinated protein by
a proteasome, a large, cylindrical
multisubunit complex
Ubiquitin
The
pathway contd
numerous proteasomes present in the
cell cytosol proteolytically cleave ubiquitintagged proteins in an ATP-dependent
process that yields peptides and intact
ubiquitin molecules
The Ubiquitin-mediated Pathway
To
be targeted for degradation by the ubiquitinmediated pathway, a protein must contain a
structure that is recognized by a ubiquitinating
enzyme complex.
Different
conjugating enzymes recognize
different degradation signals in target proteins.
– For example, the internal sequence Arg-X-X-Leu-Gly-
X-Ile-Gly-Asx in mitotic cyclin is recognized by the
ubiquitin-conjugating enzyme E1.
–Internal sequences enriched in proline, glutamic acid,
serine, and threonine are recognized by other
enzymes.
The Ubiquitin-mediated Pathway contd
The
life span of many cytosolic proteins is
correlated with the identity of the N-terminal
residue, suggesting that certain residues at the
N-terminus favor rapid ubiquitination.
–
–For example, short-lived proteins that are degraded
within 3 minutes in vivo commonly have Arg, Lys,
Phe, Leu, or Trp at their N-terminus.
–In contrast, a stabilizing amino acid such as Cys,
Ala, Ser, Thr, Gly, Val, or Met is present at the Nterminus in long-lived proteins that resist proteolytic
attack for more than 30 hours.
The Ubiquitin-mediated Pathway
contd
all
newly synthesized proteins have
methionine, a stabilizing amino acid, at
the N-terminus.
subsequent
enzymatic alteration that
generates one of the destabilizing
amino acids at the N-terminus is
necessary to target a protein for
degradation
Denaturation
Denaturation
is the breaking of the
noncovalent bonds which determine the
structure of a protein.
Complete
disruption of tertiary structure
is achieved by reduction of the disulfide
bonds in a protein.
Generally, the denatured protein will lose
its activity, antigenicity, and become
insoluble.

Denaturation
Denaturation occurs when:
–hydrogen bonds are disrupted
–disulfides are reduced
–soaps separate the hydrophobic sections
–acids or bases neutralise the salt bridges
–metals complex with functional groups to
form insoluble salts.
Denaturation
Any chemical or physical agent that
destroys and changes protein
conformations causes denaturation.
Heat
Surfactants
Urea
Reducing agents
Acids
Bases
Heavy metals UV
Alcohols
Amines
Free
radicals
Mechanisms of Denaturation
Heat:
Disrupts low energy van der Waals forces in
proteins.
Extremes
of pH: Lead to changes in the charge of
the protein’s amino acid side chains and results in
the disruption of electrostatic and hydrogen bonds.
Detergents
like Triton X-100 (nonionic, uncharged)
and sodium dodecyl sulfate (SDS, anionic, charged)
disrupt the hydrophobic forces which fold proteins.
Charged detergents like SDS also disrupt
electrostatic interactions.