ALLIED BIOCHEMISTRY II
UNIT I
Buffers: Concept of acid base indicators, buffer systems of blood and body fluids. Components
of the pH meter and the concept of pH. Chromatography: Paper, TLC, Molecular sieve and
affinity chromatography: their applications.
UNIT II
Electrophoresis:
Paper
and
Gel.
Principles
and
applications
of
colorimetry
and
spectrophotometry. Isotopes: Definition and units of radioactivity: examples of natural and heavy
isotopes in biological investigations.
UNIT III
Bioenergetics: Basic principles of thermodynamics – entropy, enthalpy and free energy; high
energy phosphates, oxidation-reduction reactions. Mitochondria: - Respiratory chain and
oxidative phosphorylation.
UNIT IV
Metabolic pathways:
Carbohydrate
metabolism:
Glycolysis,
TCA
cycle,
HMP
shunt,
Glycogenesis
and
glycogenolysis. Lipid metabolism: Beta-oxidation, biosynthesis of saturated fatty acids- Palmitic
acid.
UNIT V
Protein metabolism: General pathway of amino acid metabolism – deamination, transamination
and decarboxylation. Urea cycle. Glycine and phenylalamine metabolism (structures nor
required) Inter-relationship of carbohydrate, fat and protein metabolism (Flow chart only).
Test Books:
1. Fundamentals of Biochemistry – A.C. Deb New Central Book Agency, Calcutta 6th
Edition.
Reference Books:
1. Biochemistry – Lehninger, Nelson, Cox-CBS Publishers
2. Harper’s Biochemistry: R.K. Murray, D.K Granner, P.A. Mayes and U.W.Rodwell –
Lange Medical publications, 23rd edition.
3. Textbook of Medical Biochemistry – Rana Shindae and Chatterjee.
4. An Introduction to practical Biochemistry – D.T. Plummer.
NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF MICROBIOLOGY WITH NANOTECHNOLOGY
E-LEARNING
CLASS
: II B.Sc.
SUBJECT
: ALLIED BIOCHEMISTRY II
_________________________________________________________________________
UNIT I
Buffers: Concept of acid base indicators, buffer systems of blood and body fluids. Components
of the pH meter and the concept of pH. Chromatography: Paper, TLC, Molecular sieve and
affinity chromatography: their applications.
PART A
1. The most important buffering system for maintaining proper blood pH is:
a. the charges on the amino acids
b. the bicarbonate buffer system of CO2, carbonic acid, and bicarbonate
c. phosphate groups of serum phosphoproteins
d. none of the above
Ans: (B)
2. Myoglobin binding of oxygen depends on:
a. the oxygen concentration (pO2)
b. the hemoglobin concentration
c. the affinity of myoglobin for the O2 (K)
d. a) and c)
Ans: (d)
3. The Henderson-Hasselbalch equation:
a. allows the graphic determination of the molecular weight of a weak acid from its pH
b. does not explain the behavior of di- or tri-basic weak acids
c. employs the same value for pKa for all weak acids
d. is equally useful with solutions of acetic acid and hydrochloric acid
Ans: (C) Mitochondria
4.. The power house of the cell is
(A) Nucleus
(B) Cell membrane
Ans: (C) Mitochondria
(C) Mitochondria
(D) Lysosomes
PART B
1. Write a note on principles of operation of a pH meter
Definition
pH was defined as the negative base 10 logarithm of the hydrogen ion concentration.
pH meter
A pH meter is an electronic instrument measuring the pH (acidity or alkalinity) of a
liquid (though special probes are sometimes used to measure the pH of semi-solid substances). A
typical pH meter consists of a special measuring probe (a glass electrode) connected to an
electronic meter that measures and displays the pH reading.
A pH meter is essentially a voltmeter with a high input impedance which measures the
voltage of an electrode sensitive to the hydrogen ion concentration, relative to another electrode
which exhibits a constant voltage. pH is measured using a setup with two electrodes: the
indicator electrode and the reference electrode. These two electrodes are often combined into one
- a combined electrode.
The key feature of the pH-sensitive electrode is a thin glass membrane whose outside
surface contacts the solution to be tested. The inside surface of the glass membrane is exposed to
a constant concentration of hydrogen ions (0.1 M HCl). Inside the glass electrode assembly, a
silver wire, coated with silver chloride and immersed in the HCl solution, is called an Ag/AgCl
electrode. This electrode carries current through the half-cell reaction. The potential between the
electrode and the solution depends on the chloride ion concentration, but, since this is constant
(0.1 M), the electrode potential is also constant.
A reference electrode is needed to complete the electrical circuit. A common choice is to
use another Ag/AgCl electrode as the reference. The Ag/AgCl electrode is immersed in an 0.1 M
KCl solution which makes contact with the test solution through a porous fiber which allows a
small flow of ions back and forth to conduct the current. The potential created at this junction
between the KCl solution and the test solution is nearly zero and nearly unaffected by anything
in the solution, including hydrogen ions.
Combined electrodes with symmetrical electrode chains are the optimal construction for
obtaining temperature equality in the two electrodes. In these electrodes the inner electrode of
the glass electrode is the same type (Ag/AgCl) and has the same dimensions as the reference
electrode, and the inner solutions are as identical as possible (saturated with KCl).
Glass electrode
Reference electrode
Combined electrode
PART C
1.DISCUSS ABOUT PAPER CHROMATOGRAPHY
Chromatography was invented by M.Tswett, a botanist in 1906 in Warsaw, for the
separation of colored substances into individual components. Since then, the technique has
undergone tremendous modifications so that now a day various types of chromatograph are in
use to separate almost any given mixture, whether colored or colorless, into its constituents and
to test the purity of these constituents.Essentially, the technique of chromatography is based on
the difference in the rate at which the components of a mixture move through a porous medium
(called stationary phase) under the influence of some solvent or gas (called moving phase).
Classification of Chromatography
The moving phase may be a liquid or a gas. Based on the nature of the fixed and moving phases,
different types of chromatography are as follows
Adsorption Chromatography
It is based on the differences in the adsorption coefficients. In this the fixed phased is a solid,
e.g., alumina, magnesium oxides, silica gel, etc. The solutes are absorbed in different parts of the
adsorbent column. The adsorbed components are then eluted by passing suitable solvents
through the column.
Partition Chromatography
It is operated by mechanism analogous to counter- current distribution. In this case, the solute
gets distributed between the fixed liquid and the moving liquid (solvent). This technique is called
partition chromatography. Paper chromatography is a special case of partition chromatography in
which the madsorbent column is a paper strip.
Gas Chromatography
When the moving phase is a mixture of gases, it is called gas chromatography or vapor phase
chromatography (VPC)
Paper Chromatography
Principle
This technique is a type of partition chromatography in which the substances gets distributed
between two liquids, i.e., one is the stationary liquid (usually water) which is held is the fibers of
the paper and called the stationary phase; the other is the moving liquid or developing solvent
and called the moving phase. The components of the mixture to be separated migrate at different
rates and appear as spots at different points on the paper. It was used to separate mixture of
organic substances such as dyes and amino-acids. But now this method has been perfected to
separate cations and anions of inorganic substances.
Procedure
A drop of the test solution is applied as a small spot on a filter paper and the spot is dried. The
paper is kept in a closed chamber and the edge of the filter paper is dipped into a solvent called
developing solvent. As soon as the filter paper comes in contact with the liquid, the liquid moves
through the paper by the capillary action and when it reaches the spot of the test solution (a
mixture of 2 or more substances), the various substances are moved by solvent system at various
speeds. When the solvent has moved these cations at various speeds and suitable height (15- 18
cm) the paper is dried and various spots are visualized by suitable reagents called visualizing
reagents. The movement of substances relative to the solvent is expressed in term of RF value,
i.e., migration parameter.
Migration Parameter
The position of migrated spots on the chromatograms is indicated by the term RF.The RF is
related to the migration of the solvent front as:
Rf = Distance travelled by the solvent from the origin line
Distance travelled by the solute from the origin line
The RF Value of the substance depends upon a number of factors. They are
1. The solvent employed
2. The medium used for separation i.e., the quality of paper in case of paper
chromatography
3. The nature of the mixture
4. The temperature
5. The size of the vessel in which the operation is carried out.
Keeping the above factors constant, it is possible to compare the RF value of different
substances.
Types of Paper Chromatography
Descending chromatography
When the development of the paper is done by allowing the solvent to travel down the
paper, it is known as descending technique.
Ascending chromatography
When the development of the paper is done by allowing the solvent of travel up the
paper, it is known as ascending technique. In ascending chromatography, the mobile phase is
placed in a suitable container at the bottom of chamber. The samples are applied a few
centimeters from the bottom edge of the paper suspended from a hook.
Both ascending and descending techniques have been employed for separation of organic
and inorganic substances. But the descending technique is preferred if the RF values of various
constituent are almost same.
Radial Paper Chromatography
This is also known as circular paper chromatography. This is also known as circular
paper chromatography. This makes use of radial development. In this technique a circular filter
paper is employed.
Experimental details for qualitative analysis.
Choice of the Paper chromatographic technique.
The first job is to select the mode of paper chromatographic technique. i.e. ascending,
descending, ascending-descending, radial or 2 dimensional technique. The choice of technique
depends upon the nature of the substance to be separated. The filter paper plays an important role
in the success of paper chromatography. There are various types of Whatman chromatography
papers are available. The choice of the paper depends upon the type of separation.
Proper developing solvent
The best possible developing solvent is generally selected for the separation of substances
under examination. Commonly used solvents are, n-hexane, cyclohexane, carbon tetrachloride,
benzene, toluene, diethyl either, chloroform, ethyl acetate, n-butanol, n-propanol, acetone,
ethanol, water etc.,
Preparation of Samples
Spotting
For ascending technique, a strip of whatman filter paper of suitable size (25cm x 7cm) is
generally used. A horizontal line is drawn on the filter paper by a lead pencil. This line is known
as origin line. On the origin line, cross marks (x) are made with a pencil in such a way that each
cross (x) is at least 2 cm away from each other. With the help of a graduated micropipette, the
test solutions are applied on cross (x) marks .The spots are dried continuously by a stream of the
hot or cold air.
Drying the Chromatograms
The wet chromatograms after development are dried in special drying cabinets which are
being heated electrically with temperature controls.
Visualization
Visualization of the spots can be used in 2 ways.
(i) Chemical methods (Or)
(ii) Physical methods
(i) Chemical Detection
Chemical treatment can develop the colour of colorless solvents on the paper. The
reagents used for visualizing the spots are known as chromogenic reagents or visualizing
reagents.
(ii) Physical Methods
Some colorless spots when held under a uv lamp, fluoresce and reveal their existence.
4.6 Calculation of RF Values
The distance of chromotographed species is noted from its centre of the origin line. This distance
of solvent front from the origin lines gives the RF Values.
2. DESCRIBE THE METHOD OF THIN LAYER CHROMATOGRAPHY
Introduction
Thin layer chromatography was first discovered by Izmailov and Shraiber in 1938.
Further Stahl (1958) perfected the method and developed equipment and standardized adsorbents
for the preparation of uniform layers on glass plates. And this technique, chromatography using
thin layers of an adsorbent held on a glass plate or other supporting medium is called as thin
layer chromatography.
Preparation of thin layers on plates
Coating of glass plates with adsorbent layer is made by spreading, pouring, spraying or
dipping. The adsorbent layer may be solid or loose. For solid layers, a uniform layer of adsorbent
material could be applied to a lean glass plate with an applicator. The applicator used for the
preparation of 0.25 mm layer thickness film is the Stahl’s original applicator. Sometimes a
binder such as calcium sulphate is added to the adsorbent in small quantities before coating.
Loose layers may be prepared by dipping the plates in the suspension, spraying a thin suspension
or pouring of suspension on to the plate.
Application of sample on the chromo plates
The application of sample on the chromo plates are similar to those used in paper
chromatography. In T.L.C. 0.1 – 1 % solution of the sample are applied to the plates with the
help of capillaries, micropipettes and micro syringes. The sample solution could be applied as
single sports in a row along one side of the plate, about 2 cms from the edge.
Choice of adsorbent
The commonly used adsorbents in TLC are silica gel, alumina, Kieselguhr and powdered
cellulose. Silica gel is the most widely used adsorbent. It is slightly acidic in nature. Alumina is
basic, and Kieselguhr is a natural adsorbent.Layers of about 0.25 mm thick can be prepared by
spreading aqueous slurry of the adsorbent with a commercial applicator on glass plates. Thick
layers are air dried for about ten minutes and then activated by heating in an oven at 1100 C for 2
hours.
Choice of solvents
The choice of solvent depends on the nature of substances to be separated and the
material on which the separation is to be carried out. Polar solvents are preferred, because it
results in better separation. And a combination of two solvents too gives better results. Hence the
solvent mixture which is highly preferred for its efficiency is n-hexane-diethyl ether acetic acid
in the ratio 90:10:1.
Developing chamber
The thin layer chromatoplates are usually developed by placing them on edges in the jar
containing a 0.5 – 10 cm layer of the solvent. The jar is then covered with an air tight lid. After
the developing solvent ascends for about 10-12 cm above the origin, the plate is taken out of the
jar.
Detecting reagents.
Detecting reagents used in paper chromatography may be used in T.L.C. also. Iodine
dissolved in an organic solvent could be sprayed to deduct many components. Sulphuric acid
also forms complex which are colored and visible in day and all light.
Development and detection
The various development techniques available include ascending development, horizontal
development, multiple development, stepwise development, gradient development, continuous
development and two dimensional development. Normally ascending development is commonly
used. During ascending development the sample is spotted at one end of the plate and then
developed by ascending technique. The plates are usually placed vertically in a container
saturated with developer vapor and solvent.
Types of TLC
Adsorption TLC: Scientist Kucharezyk (1963) has used adsorption TLC for the fractionation and
analysis of petroleum products. Further it has been used for analysis of waxes and fats, for
analysis of essential oils, analysis of carotenoids, steroids, fat soluble vitamins and certain
alkaloids by different scientists.
Ion exchange TLC: This type of TLC has been used for the separation of ionic compounds from
non ionic compounds. It has been used for the fractionation of short chain carboxylic acids,
sugars, amino acids, nucleotides and detergents.
The other types of TLC include the Partition T.L.C. and Reverse phase partition T.L.C.
Advantages of TLC

The technique helps in the separation of even microgram of the substances.

The separation is very sharp.

It is used to study various biological changes, to study fractionation of large

number compounds, to analyze urine and blood samples.
UNIT II
Electrophoresis: Paper and Gel. Principles and applications of colorimetry and
spectrophotometry. Isotopes: Definition and units of radioactivity: examples of natural and heavy
isotopes in biological investigations.
______________________________________________________________________________
PART A
1. The four nucleotides are adenine (A), cytosine (C), guanine (G) and thymine (T).
(a) True
(b) False
Ans: (a) True
2. Scientists synthesize fragments of DNA and RNA using a process known as polymerase
chain reaction (PCR).
(a) True
(b) False
Ans: (a) True
3. What is RNA polymerase?
(a) An enzyme used in the synthesis of RNA
(b) An enzyme used in the synthesis of ribosomes
(c) An enzyme used in the synthesis of protein
(d) An enzyme used in the synthesis of pre-DNA
Ans: (a) An enzyme used in the synthesis of RNA
4. What is the promotor site?
(a) The site where RNA polymerase binds to DNA
(b) The site where RNA polymerase binds to protein
(c) The site where RNA polymerase binds to free nucleotides
(d) The site where RNA polymerase binds to AAA
Ans: (a) The site where RNA polymerase binds to DNA
PART B
1. ANALYSE THE BASIC PRINCIPLE BEHIND COLORIMETRY
 Photometry – measurement of light absorption – measures the intensity of the color.
 Visible light / white light – VIBGYOR
 Light beam
Colored substance
Transmitted intensity less.
Beer-Lambert’s Law:
 A α C (Beer’s law)
 A α L (Lambert’s law)
 Thus, A α C x L
A = eCL, ‘e’-extinction co-efficient.
Also A = log Ii / It = log 100 / T = log 100 – log T = 2 – log T
Molar extinction co-efficient:
 Concentration 1M / L , Path of light 1cm, then ‘e’ is molar extinction co-efficient (∑M)
Specific extinction co-efficient:
 1% (W / V) solution , Path of light 1cm
 BSA = 0.667, IgG = 1.35, IgM = 1.2 at 280nm.
Limitation of Beer-Lambert’s Law:
 Deviation due to instrument: 640nm (red) – 625 to 655nm.
 Deviation due to sample: 1. threshold concentration exceeds 2. acid-base & temp, 3.
polymerization & coagulation, 4. physically adsorbed, 5. fluorescence, 6. dilution of
colored solution (e.g. Cr2O7- + H2O
2CrO4 - + 2H+)
Orange (340nm)
yellow (770nm)
3. DISCUSS THE PRINCIPLES OF COLORIMETER
Complementary colors:
s.no
1
2
3
4
5
6
7
Color
of
solution
Violet
Blue
Green
Yellow
Orange
Red
Purple
the Range
wavelength
400-465
465-482
498-530
576-580
587-610
617-660
670-720
of
Complementary color
Greenish yellow
Yellow
Red purple
Blue
Greenish blue
Bluish green
Green
Complementary wavelength:
 λ max
Experiments using colorimeter:
 To find out complementary color / wavelength.
 To verify Beer-Lambert’s law.
Calibration:
 2.3% cobalt chloride in 1% HCl.
 Take reading at 500,505,510,515,520nm – Maximum absorbency between 505 and
515nm. (Spectronic 20)
 Take reading at 510nm with stock and 1:1 dilution with 1% HCl – absorbency value
should be 1 / 2. (colorimeter)
PART C
1. GIVEN AN ACCOUNT ON ELECTROPHORESIS
When a potential difference is applied between the 2 electrodes in a colloidal solution, it
has been observed that the colloidal particles are carried to either the positive or negative
electrode. Electrophoresis may be defined as the migration of colloidal particles through a
solution under the influence of an electrical field.
The rate of travel of the particle depends upon the following factors:
2. Characteristics of the particle
3. Properties of the electric field
4. Temperature
5. Nature of the suspending medium
Electrophoresis is the motion of dispersed particles relative to a fluid under the influence
of an electric field. Electrophoresis is the most known electrokinetic phenomena. It was
discovered by Reuss in 1809. He observed that clay particles dispersed in water migrate under
influence of an applied electric field. Electrophoresis occurs because particles dispersed in a fluid
almost always carry an electric surface charge. An electric field exerts electrostatic Coulomb
force on the particles through these charges.
Types of Electrophoresis
There are two main types of electrophoretic methods, depending upon whether the
separation is carried out in the absence or presence of a supporting or stabilizing medium. When
the separation is carried out in the absence of the stabilizing medium, the method is called free
solution method, and when it is carried out in the presence of a stabilizing medium, such as paper
the technique is known as electro chromatography or zone electrophoresis.
Free solution method
This method was first proposed by Picton and Lindex (1892), but was not fully developed
until 1937. Tiselins described the apparatus and methodology for which he was awarded Nobel
Prize. In free solution electrophoresis, the sample solution is introduced at the bottom of a U tube
that has been filled with unstabilized buffer solution. The samples are usually injected into the
bottom of the U tube through a capillary tube side arm. An electrical field is applied by means of
electrodes located at the ends of the tube. The differential movement of the charged particles
towards one or the other electrode is then observed. Separation takes place as a result of
differences in mobilities. The mobility of a particle is approximately proportional to its charge to
mass ratios. The free solution method was perfected by Tiselins. He applied this method for the
separation of proteins.
Zone electrophoresis or Electro chromatography
Many of the experimental difficulties in free solution electrophoresis are avoided, if the
separations are carried out in a stabilizing medium, such as paper. Such separations are made
possible by using a supporting medium to keep convection currents from distorting the
electrophoretic pattern. The separations depend mainly upon the properties of medium and may
resultprimarily from the electrophoretic effect or from a combination of electrophoresis, and
adsorption, ion exchange or other distribution equilibria.
Paper Electrophoresis
1. The apparatus should be set up on level surface and the electrode chambers must be filled with
buffer.
2. Provision is made for adjusting the electrolyte (buffer) in the electrode chambers to equal
levels so that siphoning action does not occur through the bed, because siphoning action across
the bed will displace and distort the electrophoretic pattern.
Types of Supporting or stabilizing or stabilizing medium
The solid supporting media in electro chromatography are as numerous and varied as
found in the other chromatographic methods.
Examples of solid supporting medium are as follows.
Filter paper, cellulose acetate strips, starch powder, cellulose powder, starch gel, agar gel,
synthetic gel ion exchange resins and membranes, asbestos paper, rayon acetate cloth, glass fibre
paper, silica powder, Kieselguhr, glass powder, silica gel, agarose gel etc.
2.DISCUSS ABOUT MOLECULAR SIEVING CHROMATOGRAPHY
It’s called also Gel permeation, Molecular sieving or Molecular Exclusion, It is a
particular type of liquid-liquid chromatography (partition chromatography) used for the
separation of substances according to the differences in sizes of their molecules, or as called
according to the difference in the Relative Size of the molecule
There are different materials used in gel filtration which are as the following
A) SEPHADEX
It is a cross-linked dextran (polysaccharaid polymer), which at the macroscopical level
the product is in the form of spherical beads, because of its high content of hydroxyl groups,
it
have
great
affinity
for
water,
therefore,
it
will
swell
up
in
water
or electrolyte solutions to give semi-transparent gel particles
B) Bio-Gel Series
It is suitable for gel filtration in aqueous media, It is based on cross linked poly-acrylamide gels
C) Agarose
It
is
fractionation
a
of
neutral
polymer
substances
derived
of
really
from
high
agar,
molecular
It
is
used
weights
for
as
the
certain
polysaccharaids, proteins & nucleic acids
D) Specially Modified Media
It
hydroxyl
is
a
groups
product
with
derived
a
from
reagent
to
the
dextran-based
render
them
gels
by
hydrophobic,
reacting
The
the
modified
gel particles swell in non-aqueous solvents
A new gel, sephadex LH-20 has recently became available, some of the hydroxyl groups
of the dextran gel are alkylated so that the gel will swell in polar organic
solvents, water or mixtures of the 2 Styro-gel, it is a rigid cross-linked polysterene gel
THE PRINCIPLE OF SEPARATION
The
&
this
cross-linking
fractionation
range
in
is
turn
agent
used,
of
these
inversely
The
gels
depends
proportional
greater
the
upon
to
amount
the
the
of
pore
size
amount
of
cross-linking
agent
used so the less the swelling properties of the gel.
The usuall way of characterizing various types of gel is by means of their water regain
value (WRV). This represents the amount of water (in ml) that is retained by 1g of
the
dry
gel
grains,
The
type
numbers
of
sephadex
&
the
bio-gel
series are TEN times the WRV, sephadex G10 has a WRV of 1 & sephadex G2000 has a WRV
of 20, These values doesn’t include the water between the grains.
The types with low WRV have smaller pore size & are used for the
fractionation
molecular
sephadex
700
peptides
&
of
weight
G10
small
are
will
sephadex
with
particle
molecules,
fractionated
fractionate
high
substances
G2000
molecular
with
weights
will
while
with
fractionate
ranging
hundered thousands, other types cover intermediate ranges
from
water
types
with
high
regain
gels,
Thus
molecular
weight
globular
5000
up
proteins
up
to
to
&
several
SEPARATION TECHNIQUES
A)
The
sample
is
applied
to
the
top
of
the
column
&
then
washed
the
swollen
through the bed of the gel particles with water or buffered solution
B)
substances
beads
(above
particles
&
with
molecules
larger
than
the
largest
the
exclusion
limit)
are
not
able
therefore
pass
through
the
pores
to
of
penetrate
bed
in
the
to
varying
the
liquid
gel
phase
outside the particles & emerge from the column first
C)
Smaller
upon
their
between
smaller
molecules
penetrate
shape
size,
There
liquid
inside
the
molecules,
the
larger
the
the
&
the
particles
is
gel
the
thus
a
partition
particles
&
percentage
extents
of
that
of
depending
the
molecules
outside,
liquid
within
The
the
particles that is available to them
D)
Molecules
therefore
molecular
size,
by
smaller
the
ranges.
the
leave
larger
sizes
the
size
depending
column
will
on
leave
their
in
the
the
order
comlumn
partition
of
first
(shape
decreasing
followed
&
size)
Note the following
A)
The
through
flow
of
the
whereas
the
mobile
column
smaller
phase
un-hindered,
molecules
will
cause
without
will
be
larger
molecules
penetrating
retarded
the
to
gel
according
pass
matrix,
to
their
penetration of the gel
B) The components of the mixture thus emerge from the column in order of
relative
molecular
completely
and
excluded
similarly,
not
be
mass,
the
from
small
separated
the
largest
gel
molecules
from
each
first,
will
that
other,
not
Any
be
components
separated
completely
from
penetrate
Molecules
of
that
are
each
other,
the
gel
will
intermediate
size
will
be retarded to a degree dependent on their penetration of the matrix
APPLICATION OF GEL CHROMATOGRAPHY
A) Analysis of mixtures of molecules of different molecular weight
B)
Molecular
molecular
globular
weight
size,
investigations
proteins
on
determined
by
a
range,
considerable
function
curve
of
of
determination,
the
proteins
the
have
sephadex
Although
gel
filtration
depends
on
shown
that
elution
volumes
of
G100
their
the
logarith
of
known
G200
molecular
elution
of
&
the
volume
molecular
molecular
weight
types
are
largely
weights.
is
Over
approximately
weight,
can
If
be
a
drawn
a
linear
calibration
up,
the
molecular
weight
of
an
unknown
protein
can
be
determined,
This
kind
of
work is very valuable in enzyme work
C)
&
De-salting,
one
of
the
common
separation
small
molecules
from
macromolecules,
distribution
coeffecient
makes
it
posssible
problems
The
to
use
is
the
larger
simple
removal
differences
column
salts
in
with
high flow rate to de-salt mixtures or compounds
3.ANALYSE THE BASIC PRINCIPLES OF SPECTROPHOTOMETRY
Light is generated by a source lamp which is normally a tungsten lamp for the visible
region of the spectrum and deuterium for the ultra-violet range. The light is dispersed into its
constituent wavelengths in a monochromator which results in a narrow band of the dispersed
spectrum passing from the exit slit of the monochromator. Suitable optics are used to lead this
light, of a narrow wavelength band, to the sample to be measured. A sample with a UV/Visible
chromophore sample absorbs a certain amount of light and the remaining light is detected by a
suitable detector in the spectrophotometer.
The Beer-Lambert law is then applied to determine the concentration of a specific analyte in the
sample at a specific wavelength:
A=εxlxc
where, at a specific wavelength, A is the measured absorbance, ε is the molar absorptivity or
extinction coefficient (M-1 cm-1), l is the path length (cm), c is the analyte concentration (M).
The relationship between Absorbance andTransmittance is:
A = log T
The Beer-Lambert law describes the linear relationship between absorbance and concentration.
However, there are some restrictions to the law, and the linearity of the Beer-Lambertl aw is
limited by chemical and instrumental factors.
Causes of non-linearity include:
• deviations in molar absorptivity coefficients at high concentrations (>0.01M) due to
electrostatic interactions between molecules in close proximity.
• scattering of light due to particulates in the sample.
• fluorescence or phosphorescence of the sample.
• stray light (i.e. light other than light of the selected wavelength reaching the detector).
• non-monochromatic radiation.
• changes in refractive index at high analyte concentration.
• shifts in chemical equilibria as a function of concentration.
• very large and complex molecules. However in practice, provided that steps are taken to ensure
that the concentration is measured in the linear part of the calibration function, the Beer-Lambert
law applies.
UNIT III
Bioenergetics: Basic principles of thermodynamics – entropy, enthalpy and free energy; high
energy phosphates, oxidation-reduction reactions. Mitochondria: - Respiratory chain and
oxidative phosphorylation.
______________________________________________________________________________
PART A
1) The term anaerobic means
A) without bacteria.
B) without CO2.
C) without ATP.
D) without O2
Ans: D) without O2
2) How do cells capture the energy released by cellular respiration?
A) They store it in molecules of carbon dioxide.
B) They produce glucose.
C) The energy is coupled to oxygen.
D) They produce ATP.
Ans: C) The energy is coupled to oxygen.
3) Which one of the following is true?
A) Cellular respiration occurs in mitochondria and in chloroplasts.
B) Photosynthesis occurs in chloroplasts and cellular respiration occurs in mitochondria.
C) Photosynthesis occurs in mitochondria and in chloroplasts.
D) Photosynthesis occurs in mitochondria and cellular respiration occurs in chloroplasts.
And: B) Photosynthesis occurs in chloroplasts and cellular respiration in mitochondria.
4) Respiration _____, and cellular respiration ______.
A) uses glucose . . . produces glucose
B) produces glucose . . . produces oxygen
C) is gas exchange . . . produces ATP
D) produces ATP . . . is gas exchange
Ans: D) produces ATP . . . is gas exchange
5) Which of the following are products of cellular respiration?
A) energy to make ATP and carbon dioxide
B) glucose and carbon dioxide
C) oxygen and energy to make ATP
D) oxgyen and carbon dioxide
Ans: C) oxygen and energy to make ATP
PART B
1.BRING OUT THE STEPS OF OXIDATION-REDUCTION REACTIONS
Oxidation is a chemical change in which electrons are lost by an atom or group of atoms, and
reduction is a chemical change in which electrons are gained by an atom or group of atoms.

Oxidation is loss of electrons

Reduction is gain of electrons
Oxidizing and reducing agents
In redox processes the reductant transfers electrons to the oxidant. Thus, in the reaction,
the reductant or reducing agent loses electrons and is oxidized, and the oxidant or oxidizing
agent gains electrons and is reduced. The pair of an oxidizing and reducing agent that are
involved in a particular reaction is called a redox pair.
Oxidizers
Substances that have the ability to oxidize other substances are said to be oxidative or
oxidizing and are known as oxidizing agents, oxidants, or oxidizers. The oxidant removes
electrons from another substance, and is thus itself reduced. And, because it "accepts" electrons,
it is also called an electron acceptor. Oxidants are usually chemical elements or substances with
elements in high oxidation numbers (e.g., H2O2, MnO4, CrO3, OsO4) or highly electronegative
substances/elements that can gain one or two extra electrons by oxidizing an element or
substance (O, F, Cl, Br).
Reducers
Substances that have the ability to reduce other substances are said to be reductive or
reducing and are known as reducing agents, reductants, or reducers. That is, the reductant
transfers electrons to another substance, and is thus itself oxidized. And, because it "donates"
electrons, it is also called an electron donor. Electron donors can also form charge transfer
complexes with electron acceptors. Electropositive elemental metals, such as lithium, sodium,
magnesium, iron, zinc, aluminium, carbon, are good reducing agents.
Examples
1.
The reaction between hydrogen and fluorine is an example of an oxidation-reduction reaction:
H2 + F2 → 2 HF
The overall reaction may be written as two half-reactions:
H2 → 2 H+ + 2 e− (the oxidation reaction)
F2 + 2 e− → 2 F− (the reduction reaction)
2.
The oxidation of iron(II) to iron(III) by hydrogen peroxide in the presence of an acid:
Fe2+ → Fe3+ + e−
H2O2 + 2 e− → 2 OH−
Overall equation:
2 Fe2+ + H2O2 + 2 H+ → 2 Fe3+ + 2 H2O
PART C
1. DEFINE THERMODYNAMICS? BRING OUT THE SIGNIFICANCE .
Thermodynamics is the science of energy conversion involving heat and other forms of energy,
most notably mechanical work.
Thermodynamics: thermo = heat (energy)
dynamics = movement, motion
Thermodynamics defines four laws which do not depend on the details of the systems
under study or how they interact. Hence these laws are generally valid and can be applied to
systems about which one knows nothing other than the balance of energy and matter transfer.
These four laws are:
Laws of thermodynamics
Zeroth law of thermodynamics: If two systems are in thermal equilibrium with a third, they are
also in thermal equilibrium with each other.
First Law of Thermodynamics: The total amount of energy (and mass) in the universe is
constant.
In any process energy can be changed from one form to another; but it can never be created nor
destroyed.
Second Law of Thermodynamics: In any spontaneous process the entropy of the universe
increases
ΔS universe = ΔS system + ΔSsurroundings
Second Law (variant): in trying to do work, you always lose energy to the surroundings.
Neither entropy (ΔS) or enthalpy (ΔH) alone can tell us whether a chemical reaction will be
spontaneous or not.
Third law of thermodynamics: As a system approaches absolute zero, all processes cease and
the entropy of the system approaches a minimum value.
Energy: "The capacity to do work and/or transfer heat"
Forms of Energy:
Kinetic (Ekinetic = ½mv2)
Potential
Heat
Light (Electromagnetic)
Electricity
Chemical
Nuclear
Matter (E = mc2)
WORK
Enthalpy (Heats) of Reaction
The amount of heat released or absorbed by a chemical reaction at constant pressure (as
one would do in a laboratory) is called the enthalpy or heat or reaction. We use the symbol ΔH
to indicate
enthalpy.
+ΔH indicates that heat is being absorbed in the reaction (it gets cold) endothermic
−ΔH indicates that heat is being given off in the reaction (it gets hot) exothermic
Standard Enthalpy = ΔH° (° is called a “not”)
Standard Enthalpy of Formation -- ΔH f
The amount of heat absorbed (endothermic) or released (exothermic) in a reaction in which one
mole of a substance is formed from its elements in their standard states, usually at 298 K (25°C).
Also called heat of formation.
ΔH f° = 0 for any element in its standard state (the natural elemental form at 1 atm or 1 M) at 298
K.
2H2 (g) + O2 (g)
2H2O (g)
ΔHf (H O) = 241.8 kJ/mol
Entropy
The final state of a system is more energetically favorable if:
1. Energy can be dispersed over a greater number and variety of molecules.
2. The particles of the system can be more dispersed (more disordered).
The dispersal of energy and matter is described by the thermodynamic state function
entropy, S. The greater the dispersal of energy or matter in a system, the higher is its entropy.
The greater the disorder (dispersal of energy and matter, both in space and in variety) the higher
the entropy. Adding heat to a material increases the disorder.
Ice - well ordered structure
water more disordered
water vapours most
disordered
Unlike ΔH, entropy can be defined exactly because of the Third Law of Thermodynamics:
Third Law of Thermodynamics: Any pure crystalline substance at a temperature of absolute zero
(0.0 K) has an entropy of zero (S = 0.0 J/K•mol).
+ΔS indicates that entropy is increasing in the reaction or transformation (it's getting more
disordered -- mother nature likes)
−ΔS indicates that entropy is decreasing in the reaction or transformation (it's getting less
disordered {more ordered} -- mother nature doesn't like, but it does happen)
Qualitative "Rules" About Entropy:
1) Entropy increases as one goes from a solid to a liquid, or more dramatically, a liquid to a gas.
2) Entropy increases if a solid or liquid is dissolved in a solvent.
3) Entropy increases as the number of particles (molecules) in a system increases
4) The Entropy of any material increases with increasing temperature
5) Entropy increases as the mass of a molecule increases
6) Entropy is higher for weakly bonded compounds than for compounds with very strong
covalent
bonds
7) Entropy increases as the complexity (# of atoms, # of heavier atoms, etc.) of a molecule
increases
Entropy of Methane S° = 186 J/K•mol
Entropy of Ethylene S° = 220 J/K•mol
Gibbs Free Energy
The combination of entropy, temperature and enthalpy explains whether a reaction is
going to be spontaneous or not. The symbol ΔG is used to define the Free Energy of a system.
Since this was discovered by J. Willard Gibbs it is also called the Gibbs Free Energy. "Free"
energy refers to the amount of energy available to do work once you have paid your price to
entropy.
Note that this is not given simply by ΔH, the heat energy released in a reaction.
ΔGº = ΔHº − TΔSº
When ΔG is negative, it indicates that a reaction or process is spontaneous. A positive ΔG
indicates a non-spontaneous reaction.
ΔG = ΔH – TΔS
ΔS
Δ G = negative
spontaneous
at all temperatures
Δ G = ??
Spontaneous at
low temperatures
0
Δ G = ??
spontaneous
at high temperatures
+
+

Spontaneous = exoergic (energy releasing)

Non-spontaneous = endoergic (energy releasing)
ΔH
Δ G = positive
non-spontaneous
at all temperatures
Remember that entropies are given in units of J/K•mol while enthalpies and free energies are
inkJ/mol.
2. ANALYSE THE STEPS INVOLVED IN OXIDATIVE PHOSPHORYLATION
Oxidative phosphorylation is a metabolic pathway that uses energy released by the
oxidation of nutrients to produce adenosine triphosphate (ATP).
Movement of electrons throught the electron transport system (ETS) causes protons to
be pumped from the mitochondrial matrix of a eukaryotic cell to the intermembrane space. The
difference in potential created by movement of the charged protons as well as the concentration
gradient created by the pumping provides the energy source for making ATP in the
mitochondrion. This process is called oxidative phosphorylation. It occurs as a result of protons
moving through Complex V.
The efficiency of oxidative phosphorylation is determined by the P/O ratio, which is a
measure of the amount of ATP made versus the amount of oxygen consumed
The actual site in the mitochondrion where ATP is made is Complex V (also called ATP
synthase or the F0F1 complex). It is located on the inner mitochondrial cristae.
The F0F1 Complex
Complex V (also called ATP synthase or the F0F1 complex) is a multi-protein structure
with three-fold symmetry, resembling a mushroom. It consists of a top knob called F1 and a
stalk, which joins the knob to the base called F0 in the inner mitochondrial membrane. The F1
knob projects into the mitochondrial matrix and contains three
dimers arranged like segments
of an orange around the stalk. The stalk contains and proteins; is attached to protein b of the
F0 base, which also contains proteins a and c plus others. The
stator.
abc complex is called a
Electron Transport
An electron transport chain (ETC) couples electron transfer between an electron donor
(such as NADH) and an electron acceptor (such as O2) to the transfer of H+ ions (protons) across
a membrane. Passage of the electrons through the system generates potential energy that is used
to make ATP in oxidative phosphorylation.
Above flow chart lists the contents of the various multiprotein complexes described below:
NADH and NADH Dehydrogenase (Complex I) - NADH is generated by numerous
dehydrogenases in the cell. NADH is reoxidized to NAD+ by complex I of the mitochondria
(also called NADH dehydrogenase). NADH dehydrogenase contains flavin mononucleotide
(FMN) as a tightly bound prosthetic group and catalyzes the following reaction:
NADH + H+ + FMN <=> NAD+ + FMNH2
Complex I contains about 25 separate polypeptide chains. It also contains iron-sulfur
centers, which transfer electrons from FMNH2 to the next carrier, coenzyme Q. Complex I is
also called NADH-coenzyme Q reductase because the electrons are used to reduce coenzyme Q.
Complex II (succinate dehydrogenase) - Complex II is not in the path traveled by
electrons from Complex I. Instead, it is a point of entry of electrons from FADH2 produced by
the enzyme succinate dehydrogenase in the citric acid cycle. Both complexes I and II donate
their electrons to the same acceptor, coenzyme Q. Complex II, like complex I, contains ironsulfur proteins, which participate in electron transfer. It is also called succinate-coenzyme Q
reductase because its electrons reduce coenzyme Q.
Coenzyme Q (CoQ) - CoQ is a benzoquinone linked to a number of isoprene units
(usually 10 in mammalian cells and 6 in bacteria). The isoprenoid tail gives the molecule its
apolar character, which allows CoQ to diffuse rapidly through the inner mitochondrial
membrane. CoQ has the ability to accept electrons in pairs and pass them one at a time through a
semiquinone intermediate to Complex III. This cycle is referred to as the Q cycle.
Complex III - Complex III contains a diversity of electron carrying proteins. They
include cytochrome b, iron sulfur centers, and cytochrome c1. Cytochrome b is the first of the
heme-carrying proteins involved in electron transport.
Cytochrome c - This small protein is the only one from the electron transport system not
in a complex. It accepts electrons from complex III and shuttles them to complex IV.
Complex IV - Complex IV is also known as cytochrome oxidase, because it takes
electrons from cytochrome c. Complex IV contains cytochromes a and a3. Cytochromes a and a3
evidently represent two identical heme A moieties, attached to the same polypeptide chain. They
are within different environments in the inner membrane, however, so they have different
reduction potentials. Each of the hemes is associated with a copper ion, located close to the heme
iron.
UNIT IV
Metabolic pathways:
Carbohydrate
metabolism:
Glycolysis,
TCA
cycle,
HMP
shunt,
Glycogenesis
and
glycogenolysis. Lipid metabolism: Beta-oxidation, biosynthesis of saturated fatty acids- Palmitic
acid.
______________________________________________________________________________
UNIT 1
PART – A
1. Glucose-6-phosphatase is not present in
(A) Liver and kidneys
(B) Kidneys and muscles
(C)Kidneys and adipose tissue
(D) Muscles and adipose tissue
Ans: (B) Kidneys and muscles
2. Pyruvate carboxylase is regulated by
(A) Induction
(B) Repression
(C) Allosteric regulation
(D) All of these
Ans: (D) All of these
3. Fructose-2, 6-biphosphate is formed by the action of
(A) Phosphofructokinase-1
(B) Phosphofructokinase-2
(C) Fructose biphosphate isomerase
(D) Fructose-1, 6-biphosphatase
Ans: (B) Phosphofructokinase-2
4. Galactose is phosphorylated by galactokinase to form
(A) Galactose-6-phosphate
(B) Galactose-1, 6 diphosphate
(C) Galactose-1-phosphate
(D) All of these
Ans: (B) Galactose-1, 6 diphosphate
5. Two important byproducts of HMP shunt are
(A) NADH and pentose sugars
(B) NADPH and pentose sugars
(C) Pentose sugars and 4 membered sugars
(D) Pentose sugars and sedoheptulose
Ans: (B) NADPH and pentose sugars
PART C
1. EXPLAIN SCHEMATICALLY THE PATHWAY OF GLYCOLYSIS
Glycolysis is a central metabolic pathway involving metabolism of the sugar glucose.
The glucose being divided into a phase in which ATP energy is invested and a phase in which
ATP energy is generated .The starting point glycolysis is the molecule glucose and the process
ends with formation of two pyruvate molecules. Additional products of glycolysis include two
ATPs and two NADHs.
Reactions of Glycolysis:
There are ten steps to glycolysis.
Reaction #1
α -D-Glucose + ATP <=> α -D-Glucose-6-Phosphate + ADP +H+
Enzyme: Hexokinase
ATP energy is used. Hexokinase is capable of phosphorylating glucose and making
glucose-6-phosphate, but the product of the reaction can reach high enough concentration to
inhibit hexokinase and limit glycolysis.
Reaction #2
α -D-glucose-6-phosphate <=> D-fructose-6-phosphate
Enzyme: Phosphoglucoisomerase
This is an aldose-ketose isomerization that proceeds through an enediol intermediate.
G6P is the aldose and fructose-6-phosphate is the ketose. Phosphoglucoisomerase,which
catalyzes this isomerization.
Reaction #3
D-fructose-6-phosphate + ATP <=>D-fructose-1, 6-bisphosphate
Enzyme: Phosphofructokinase
ATP energy is used to phosphorylate F6P to fructose-1, 6-bisphosphate .This reaction is
the key to understanding how regulation of glycolysis is regulated. The enzyme,
phosphofructokinase , is allosterically regulated by AMP (on), ADP (on), ATP (off), citrate (off),
and fructose-2,6-bisphosphate (on). Consequently, the reaction is essentially irreversible in vivo.
At this point all of the energy inputs for glycolysis are complete.
Reaction #4
D-fructose-1, 6-bisphosphate <=> Dihydroxyacetone phosphate + D- Glyceraldehyde-
3- Phosphate
Enzyme:
Fructose-1,
6-Bisphosphate
Aldolase
In this reaction, F1, 6BP is cleaved to yield two three-carbon intermediates,
glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).
Reaction #5
Dihydroxyacetone phosphate <=>D-Glyceraldehyde-3-Phosphate
Enzyme: Triose Phosphate Isomerase
The isomerization of DHAP to G3P, like the isomerization of G6P to F6P (reaction 2
above), proceeds through an enediol intermediate. This reaction marks the end of what is
referred to as the energy investment phase, although the last ATP energy was used in reaction
Reaction #6
D-Glyceraldehyde-3-Phosphate + NAD+ + Pi <=>1, 3 bisphosphoglycerate + NADH
Enzyme: Glyceradehyde-3-Phosphate Dehydrogenase
+ H+
In this reaction, G3P is phosphorylated and oxidized, so something (NAD+) must be
concomitantly reduced. As a result, the NAD+/NADH balance in the cell is important. If the
concentration of NAD+ is low, the reverse reaction is favored, preventing glycolysis from
occurring aerobically. Instead it must occur anaerobically. Thus this reaction determines whether
glycolysis occurs aerobically or anaerobically. 1,3-bisphosphoglycerate (1,3BPG) contains an
acylphosphate group which
phosphorylation.
is capable of synthesizing ATP via a substrate-level
In the cell, however, the forward reaction is favored, because high
NAD+/NADH ratio normally present. Note also that the NADH produced in this reaction can be
used to make three molecules of ATP in aerobic glycolysis (when oxidative phosphorylation is
occurring).
Reaction #7
1, 3 bisphosphoglycerate + ADP <=> 3-phosphoglycerate + ATP
Enzyme: Phosphoglycerate Kinase
This reaction is a substrate-level phosphorylation of ADP to produce 3-phosphoglycerate
(3PG) and the first ATP of glycolysis. Because two molecules of ATP are produced per molecule
of glucose, the net yield of ATP is zero at this stage of glycolysis.
Reaction #8
3-phosphoglycerate <=> 2-phosphoglycerate
Enzyme: Phosphoglycerate Mutase
Formation of 3PG over 2PG ocurs under standard conditions, but in the cell the
concentration of 3PG is kept high relative to the concentration of 2PG, which drives the reaction
to the right.
Reaction #9
2-phosphoglycerate <=> Phosphoenolpyruvate + H2O
Enzyme: Enolase
This reaction is a simple dehydration (or elimination) of 2PG to form
phosphoenolpyruvate (PEP). This high free energy of hydrolysis is necessary for the next step in
glycolysis, which is another substrate level phosphorylation of ADP to form ATP.
Reaction #10
Phosphoenolpyruvate + ADP + H+ <=> Pyruvate + ATP
Enzyme: Pyruvate Kinase
This reaction is important for several reasons. First, it generates ATP from the substratelevel phosporylation of ADP, putting the balance for glycolysis at a net gain of two molecules of
ATP per molecule of glucose. Second, it is very favorable energetically, serving to "pull" the two
preceding reactions forward. Third, the enzyme catalyzing the reaction, pyruvate kinase, is
allosterically inactivated by ATP, alanine, and acetyl-CoA, allosterically activated by F1,6BP,
and is inactivated by covalent modification (phosphorylation) from the kinase cascade.
2. EXPLAIN CITRIC ACID CYCLE.
The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the
Krebs cycle ,is a series of enzyme-catalysed chemical reactions of central importance in all
living cells that use oxygen as part of cellular respiration. In eukaryotes, the citric acid cycle
occurs in the matrix of the mitochondrion. The components and reactions of the citric acid cycle
were established by seminal work from both Albert Szent-Györgyi and Hans Krebs.
In aerobic organisms, the citric acid cycle is part of a metabolic pathway involved in the
chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to
generate a form of usable energy. Other relevant reactions in the pathway include those in
glycolysis and pyruvate oxidation before the citric acid cycle, and oxidative phosphorylation
after it. In addition, it provides precursors for many compounds including some amino acids and
is therefore functional even in cells performing fermentation.
STEPS:
Two carbons are oxidized to CO2, and the energy from these reactions is transferred to
other metabolic processes by GTP (or ATP), and as electrons in NADH and QH2. The NADH
generated in the TCA cycle may later donate its electrons in oxidative phosphorylation to drive
ATP synthesis; FADH2 is covalently attached to succinate dehydrogenase, an enzyme
functioning both in the TCA cycle and the mitochondrial electron transport chain in oxidative
phosphorylation. FADH2 thereby facilitates transfer of electrons to coenzyme Q, which is the
final electron acceptor of the reaction catalyzed by the Succinate:ubiquinone oxidoreductase
complex, also acting as an intermediate in the electron transport chain.The citric acid cycle is
continuously supplied with new carbons in the form of acetyl-CoA, entering at step 1 below.
3.DISCUSS ABOUT THE PENTOSE PHOSPHATE PATHWAY
4. DESCRIBE THE GLYCOGENESIS AND GLYCOGENOLYSIS PROCESS
GLYCOGENESIS – SYNTHEIS OF GLYCOGEN FROM GLUCOSE
GLYCOGENOLYSIS – BREAKDOWN OF GLYCOGEN TO GLUCOSE
GLYCOGENESIS AND GLYCOGENOLYSIS
5. GIVEN AN DETAILED ACCOUNT ON FATTY ACID BIOSYNTHESIS
Fatty acid biosynthesis is similar in all known prokaryotes and eukaryotes. In
eukaryotes, the biosynthesis of a fatty acid such as palmitate (C16) occurs in the cytoplasm.
The basic strategy includes the following three possible steps:
1) Synthesis of Palmitate from Acetyl-CoA
2) Chain Elongation of Palmitate (long chain fatty acids)
3) Fatty Acid Desaturation
Though the reactions in fatty acid biosynthesis resemble the reversal of the analogous
reactions in oxidation, fatty acid synthesis is distinct from fatty acid oxidation. For example,
acyl groups are carried by acyl carrier protein in fatty acid synthesis, instead of coenzyme A.
Furthermore, reducing equivalents come from NADPH and energy is provided by ATP.
Overall, the biosynthesis of palmitate from 8 acetyl-CoAs requires 7 ATPs and 14 NADPHs.
Most of the enzymatic activities required for the synthesis of palmitate from acetyl-CoA are
found on a multienzyme complex called fatty acid synthase that is composed of two
polypeptidechains.
Synthesis of Palmitate from Acetyl-CoA
Fatty acid biosynthesis from acetyl-CoA to palmitate involves an enzyme complex
called fatty acid synthase, which appears to operate by a swinging arm mechanism involving
the growing fatty acyl group linked to acyl carrier protein. Each of the individual enzymatic
activities below is a part of the fatty acid synthase complex. The first step in the synthesis of
palmitate is the synthesis of malonyl-CoA from acetyl-CoA and HCO3. This reaction
requires ATP and is catalyzed by acetyl-CoA carboxylase. It is the point of regulation of the
pathway.
Acetyl-CoA Carboxylase
Acetyl-CoA carboxylase is the primary regulatory enzyme in fatty acid biosynthesis.
The active form of the enzyme is a long filamentous array of monomer units. Monomeric
units can be either phosphorylated or dephosphorylated. Acetyl-CoA carboxylase can be
phosphorylated by two kinases, cAMP-dependent protein kinase or AMP-dependent protein
kinase.
Fatty Acid Biosynthesis
The enzyme catalyzes the addition of a carboxyl group from bicarbonate to acetylCoA, forming malonyl-CoA.
Acetyl-CoA + ATP + HCO3- <=> Malonyl-CoA + ADP + Pi + H+
Like other carboxylases, acetyl-CoA carboxylase contains a biotin cofactor.
The active form of acetyl-CoA carboxylase is a long filamentous array of monomer
units. The individual monomers are generally inactive. The malonyl-CoA produced in this
reaction, along with acetyl-CoA, provide substrates for the fatty acid synthase complex. The
various enzymatic activities of the fatty acid synthase complex are summarized as follows.
1. Acetyl-CoA + ACP <=> Acetyl-ACP + CoASH
Acetyl-CoA-ACP Transacylase
Acetyl-CoA-ACP Transacylase is an enzyme of fatty acid biosynthesis that catalyzes the
transfer of acyl carrier protein (ACP) to acetyl-CoA in the reaction below (Figure 18.24).
Acetyl-ACP is the initial two carbon unit onto which the initial malonyl-ACP is added to
form β -ketoacyl-ACP.
2. Malonyl-CoA + ACP <=> Malonyl-ACP + CoASH
Malonyl-CoA-ACP Transacylase
Malonyl-CoA-ACP transacylase catalyzes the reaction as mentioned above.
3. Acetyl-ACP + Malonyl-ACP <=> β-Ketoacyl-ACP + ACP + CO2
β-Ketoacyl-ACP Synthase
β-Ketoacyl-ACP synthase catalyzes addition of acetyl group from malonyl-ACP to
growing fatty acid chain in fatty acid biosynthesis.
4. β-Ketoacyl-ACP + NADPH + H+ <=> D-3-Hydroxyacyl-ACP + NADP+
β-ketoacyl-ACP reductase
β-Ketoacyl-ACP reductase catalyzes the reduction of β-ketoacyl-ACP
to D-3-hydroxylacyl-ACP in fatty acid biosynthesis.
5. D-3-Hydroxyacyl-ACP <=> Trans-2-enoyl-ACP + H2O
3-Hydroxylacyl-ACP Dehydrogenase
3-hydroxyacyl-ACP dehydrase is part of the fatty acid synthase complex. It catalyzes
the reaction as mentined above.
6. Trans-2-enoyl-ACP + NADPH + H+ <=> Acyl-ACP + NADP+
Enoyl-ACP Reductase
Enoyl-ACP reductase catalyzes the final reduction in the process of fatty acid
biosynthesis. Starting with acetyl-CoA, the process cycles between steps 1-6 seven times to
yield palmitoyl-ACP, which is hydrolyzed to give palmitate and ACP. Note that the CO2,
which was added to acetyl-CoA in the acetyl-CoA carboxylase-catalyzed step, is removed
subsequently and not incorporated into the final product.
6. WRITE AN ELABORATE NOTE ON Β OXIDATION OF FATTY ACIDS
Inside of the mitochondrion, fatty acyl-CoAs are oxidized in a series of steps that
each release a two-carbon fragment, in the form of acetyl-CoA. Each step involves four
reactions-dehydrogenation, hydration, dehydrogenation, and thiolytic cleavage.The
individual reactions are summarized as follows:
1.
Fatty acyl-CoA +FAD <=> Trans- 2-Enoyl-CoA + FADH2
(catalyzed by Fatty Acyl-CoA Dehydrogenase)
Fatty acyl-CoA dehydrogenase catalyzes the initial step in the process of oxidation
of fatty acids. The FAD and FADH2 in the reaction are enzyme-bound. Electrons from
FADH2 are donated to coenzyme Q in the electron transport system.The FAD and FADH2
in the reaction are enzyme-bound. Electrons from FADH2 are donated to coenzyme Q in the
electron transport system.
2. Trans- 2-Enoyl-CoA + H2O <=> L-3-Hydroxyacyl-CoA
(catalyzed by Enoyl-CoA Hydratase)
Enoyl-CoA hydratase catalyzes addition of water to trans- 2-enoyl-S-CoA in the
process of fatty acid oxidation. The product of this reaction is the stereospecific L isomer of
3-hydroxylacyl-CoA.
3. L-3-Hydroxyacyl-CoA + NAD+ <=> 3-Ketoacyl-CoA + NADH + H+
(catalyzed by 3-Hydroxyacyl-CoA Dehydrogenase)
3-Hydroxyacyl-CoA Dehydrogenase catalyzes the reaction that mentioned above.
This reaction is part of the oxidation pathway for catabolism of fatty acids.
4. 3-Ketoacyl-S-CoA + CoASH <=> Acyl-CoA + Acetyl-CoA
(catalyzed by -Ketothiolase)
- Ketotholase is an enzyme of the fatty acid oxidation cycle that adds a CoASH to
-keto-acyl-CoA to convert it to an acyl-CoA with two less carbons plus acetyl-CoA.
Thiolase is also important in formation of ketone bodies when the acetyl-CoA concentration
is high.
UNIT V
Protein metabolism: General pathway of amino acid metabolism – deamination,
transamination and decarboxylation. Urea cycle. Glycine and phenylalamine metabolism
(structures nor required) Inter-relationship of carbohydrate, fat and protein metabolism (Flow
chart only).
___________________________________________________________________________
PART A
1.Two amino acids of the standard 20 contain sulfur atoms. They are:
a. cysteine and serine
b. cysteine and threonine
c. methionine and cysteine
d. methionine and serine
Ans: c. methionine and cysteine
2. The enzyme fumarase catalyzes the reversible hydration of fumaric acid to l-malate, but it
will not catalyze the hydration of maleic acid, the cis isomer of fumaric acid. This is an
example of:
a. biological activity
b. chiral activity
c. racemization
d. stereoisomerization
Ans: d. stereoisomerization
PART B
1. WRITE A SHORT NOTE ON OXIDATIVE DEAMINATION
Deamination is also an oxidative reaction that occurs under aerobic conditions in all
tissues but especially the liver. During oxidative deamination, an amino acid is converted
into the corresponding keto acid by the removal of the amine functional group as ammonia
and the amine functional group is replaced by the ketone group. The ammonia eventually
goes into the urea cycle.
Oxidative deamination occurs primarily on glutamic acid because glutamic acid was the end
product of many transamination reactions.
The glutamate dehydrogenase is allosterically controlled by ATP and ADP. ATP acts as an
inhibitor whereas ADP is an activator.
2. WRITE A NOTE ON TRANSAMINATION
Transamination as the name implies, refers to the transfer of an amine group from
one molecule to another. This reaction is catalyzed by a family of enzymes called
transaminases. Actually, the transamination reaction results in the exchange of an amine
group on one acid with a ketone group on another acid. It is analogous to a double
replacement reaction.
The most usual and major keto acid involved with transamination reactions is alphaketoglutaric acid, an intermediate in the citric acid cycle. A specific example is the
transamination of alanine to make pyruvic acid and glutamic acid.
Other amino acids which can be converted after several steps through transamination into
pyruvic acid include serine, cysteine, and glycine.
PART C
1.DISCUSS IN BRIEF ABOUT UREA CYCLE
INTRODUCTION:
Urea is the major end product of nitrogen metabolism in humans and mammals.
Ammonia, the product of oxidative deamination reactions, is toxic in even small amounts and
must be removed from the body. The urea cycle or the ornithine cycle describes the
conversion reactions of ammonia into urea. Since these reactions occur in the liver, the urea
is then transported to the kidneys where it is excreted. The overall urea formation reaction is:
2 Ammonia + carbon dioxide + 3ATP ---> urea + water + 3 ADP
The step wise process of the urea cycle is summarized below. One amine group
comes from oxidative deamination of glutamic acid while the other amine group comes from
aspartic acid. Aspartic acid is regenerated from fumaric acid produced by the urea cycle. The
fumaric acid first undergoes reactions through a portion of the citric acid cycle to produce
oxaloacetic acid which is then changed by transamination into aspartic acid.
2. ENLIST THE IMPORTANCE OF GLYCINE BIOSYNTHESIS WITH FLOW
CHART
The main pathway to glycine is a 1-step reversible reaction catalyzed by serine
hydroxymethyltransferase (SHMT). This enzyme is a member of the family of one-carbon
transferases and is also known as glycine hydroxymethyltransferase. This reaction involves
the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF),
producing glycine and N5,N10-methylene-THF. There are mitochondrial and cytosolic
versions of serine hydroxymethyltransferase. The cytosolic enzyme is referred to as SHMT1
and the mitochondrial enzyme is SHMT2.
Glycine produced from serine or from the diet can also be oxidized by glycine
decarboxylase (also referred to as the glycine cleavage complex, GCC) to yield a second
equivalent of N5,N10-methylene-tetrahydrofolate as well as ammonia and CO2.
Glycine is involved in many anabolic reactions other than protein synthesis including the
synthesis of purine nucleotides, heme, glutathione, creatine and serine.
3. ILLUSTRATE THE INTERRELATIONSHIP OF CARABOHYDRATES, FATS
AND PROTEIN MEABOLISM WITH FLOW CHART