Biochemistry Science &

History
New Approach

Author

Abdelazeem Elhabyan

Page1
Dedicated to
My Family

Page2
About the author :
I am a Final year medical student at Tanta university who loves
biochemistry a lot and interested in medical genetics and
Genomics . I have read a lot in this field ,many textbooks
,scientific papers , attended more than 110 courses in Accredited
universities online . I am also the Regional Trainer For
Galaxyproject.org in Arabic Speaking Region and Africa and
The Founder of Arabic Community (Twitter : @Galaxy_Arabic)
,also attended conferences and workshops in many fields .
I have been invited by NHS England to attend their online
Training to Prepare future international Leaders in the Field of
Genomic Medicine . I am open science Advocate and member
of Force11 community for Open Science and Blog about
Science in HuffPost Arabi
I spend my free time writing ,chatting ,surfing the internet ,
hanging around with friends , I love Tea very much and Play
Volley Ball and Speed Ball .I love computers a lot thats what i
needed to declare . To contact me (Facebook : Habyan1 ,
Twitter : @AElhabyan,Linked In : Abdelazeem Abdelhameed ,
Email : Abdelazeem_Abdelhameed2015505@yahoo.com)

Page3
This Book is Published under Creative common License
Attribution 4.0 International (CC BY 4.0)

You are free to

Share copy and redistribute the material in any medium or format

Adapt remix, transform, and build upon the material
For any purpose, even commercially.

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Page4
List of contents
Introduction.6

Robert Hooke...9

Karl Wilhelm scheele. 9

Berzelius..10

Gerardus Johannes Mulder11

Friedrich Wohler.....11

Anselme Payen.....13

Louis Pasteur....13

Charles Edouard Brown Sequard .....19

George Mendel.21

Friedrich Miescher..24

J .Willard Gibbs25

Ludwig Boltzman28

Hermann Emil Fischer...32

Eduard Buchner.34

Sir Frederick Gowland Hopkins35

Leonor Michaelis and Maud Leonora Menten.36

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Thomas Hunt Morgan37

Theodor Svedberg .38

James B. Sumner41

Otto Warbung.42

Carl Ferdinand and Getry Cori43

Phoebus Levene..44

Albert Szent-Gyrgyi.46

Sir Hans Krebs....49

George Beadle and Edward Tatum..52

AveryMacLeodMcCarty...53

Melvin Calvin ,James Bassham and Andrew Benson.56

Edwin Chargaff...59

Linus Pauling and Robert Corey60

Alfred Hershey and Martha Chase....62

Watson and Crick63

Frederick sanger.66

Earl sutherland....67

Matthew Meselson and Franklin Stahl.68

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John Kendrew and Max Perut..71

Jens Christian Skou.....73

Ornstein and Davis..75

Charles Loe, Audrey Stevens and jerald Hurwitz....77

Francois Jacob and J. Monod.....78

Jean-pierrre Changuex , F. jacob , and J Monod 81

David Chilton Philips..83

Robert Holley...84

Jerome Vinograd.85

Hamilton Smith87

Howard temin and David Baltimore89

Stanley Cohen and Herbert Boyer90

Cesar Milsein, Georges Kohler, and Niels Kai Jerne..91

Phillip Sharp and Richard Roberts93

Karry B.Mullis.95

Refrences..98

Page7
Introduction

Chance played a role in a scientific discovery .

Szent Gyorgi wrote in his autobiography (one night , my wife

served me fresh red paprika for supper . I did not feel like eating
it so I thought of a way out. Suddenly it occurred to me that this
is the one plant I had never tested. I took it to the laboratory ...
[and by] about midnight I knew that it was a treasure full of
vitamin C.)

Cooperation between scientists

As everybody Knows science is not the effort of One scientist at

one place . it is on the contrary , a collaborated effort of many
scientists . this appears in letter sent by Berzelius to Gerardus
Mulder, where berzelius suggests a name for the discovery of
Gerardus Mulder.

"The word protein that I propose to you . . . I would wish to

derive from proteios, because it appears to be the
primitive or principal substance of animal nutrition that
plants prepare for the herbivores, and which the latter then
furnish to the carnivores. "

J. J. Berzelius,1838

Another letter from a student(Wohler) to his Mentor(Berzelius)

"I must tell you that I can make urea without thereby needing to
have kidneys, or anyhow, an animal, be it human or dog".

You will also find that a scientist work might not be appreciated
at first , it might be burried for years untill someone cares about

Page8
and rediscovers it. eg. Mendels Work which was not discovered
untill 1900.Now Mendle is considered the Father of Genetics.

Whatever the obstacles you find , you should believe in your

goal this helped James Summer who isolated the first enzyme.
Although his work was unsuccessful at first and his colleagues
were doubtful believing that what he was trying to achieve is
impossible, He did it at last.

To have your own sight is important . this is what Sutherland

proved.
"When I first entered the study of hormone action, some
25 years ago, there was a widespread feeling among
biologists that hormone action could not be studied
meaningfully in the absence of organized cell structure.
However, as I reflected on the history of biochemistry, it
seemed to me there was a real possibility that hormones
might act at the molecular level. "

Earl W. Sutherland, Nobel Address, 1971

Finally the role of science is the Good of humanity this is what

true scientist looks for.

Do we care about these people that are HIV-positive whose lives

have been ruined? Those are the people I'm the most concerned
about. Every night I think about this.

Kary Mullis(PCR Inventor)

Page9
Some scientists are followed by a short introduction about
their discovery.

Other scientists are followed by Quotes either written by

them or by other scientists but related to their discovery.

The book is ordered historically however in rare cases I

might leave the historical order to complete the discoveries
by the same scientist which occurred at later time . eg Emil
Fisher , Sir Hans Krebs and pasteur.

Each scientist with his photo and his discovery and if there
is relation between a scientist and another it is mentioned
most of times in boxes.

The date of discovery is written in Bold line like this 1836

If a scientist got Noble Prize it is mentioned at the end .

There are boxes in some pages : their content help to

simplify , add more information or link achievements of
different scientists .
Colored diagrams are also provided whenever needed to
make content clear.

Finally , Any suggestions , remarks or criticism will be greatly

welcomed , heartily appreciated and very much considered.
E-mail : Abdelazeem_Abdelhameed2015505@yahoo.com

P a g e 11
 There were later on
advancements in
micrscopic techniques
which made it possible to
determine that the cell is
the functional and
structural unit of an
Bottle cork : organism first
round piece of Leeuvenhook : made a
substance that is Microscope that
pushed into the
magnified more than that
top of a bottle to
of Robert hook and
close it.
examined different parts
of animals.
Schelden: he was the
founder of cell theory
proposing that cell is the
building block of an
In 1665 using the first microscope Robert Hooke plants.
discovered multible tiny pores on examining very
Schwan: found that all
thin slices of bottle cork that he named "cells". animal tissues are
This comes from the Latin word Cella, meaning a composed of cells.
small room. these were actually empty cell Virschow: stressed on that
walls(cellulose) of plant tissues. With microscopes cell is not only building
during this time having a low magnification, unit but also functional
unit of an organism and
Hooke was unable to see that there were other that new cells come from
internal components to the cells he was observing. older cells.
So he did not think the "cellulae" were alive. Robert Brown : he
discovered the Nucleues
and found that it contains
the hereditary materia
which pass on from
generation to another.
This is more related to
Histology but cannot be
overlooked in
Biochemistry as it is life
on cellular level.

In1781,he Isolated citric acid, lactic acid, malic

acid and Glycerol.

P a g e 11
When he treated the pyrolusite with hydrochloric acid over a
warm bath, a yellow-green gas with a strong odor was produced
This gase was chlorinewhich was later used as disinfectant in
the form of sodium Hypochlorite.

Berzelii park Stockholm Sweden

He introduced new terms to Chemistry which were later useful

in the field of Biochemistry. eg Catalyst , Polymer and Isomer .
He used the word polymer to describe organic compounds
which shared identical formulas but differ in their Molecular
Weight . He used the word to describe Glucose which he
considered a Polymer of Formaldhyde (CHO).
Also He was the first person to make the distinction between
organic compounds (those containing carbon), and inorganic
compounds. and thought that organic compounds are only
formed in living creatures which was proven to be wrong
hypothesis by his student Wohler.

P a g e 12
 The nature of coffee was not
Herbivores are animals that determined untill later by Emil
only eat plant. Fischer when he discovered
Carnivores are animals that eat Purines.
meat
Furnish : to provide someone
with something they need. The common radical of protein
synthesis was discovered later to
be Amino acids.

He earned a medical degree then started to study chemistry and

became a professor at Rotterdam University.
he started to study organic compounds like coffee and protein
.He claimed that proteins are made up of a common radical,
protein, and that protein had the same formula except for some
variation in amounts of sulfur and phosphorus.
Berzelius sent him letter and named the compounds that Mulder
examined , (Proteins ).

The word protein that I propose to you . . . I would wish to

derive from proteios, because it appears to be the
primitive or principal substance of animal nutrition that
plants prepare for the herbivores, and which the latter then
furnish to the carnivores.

J. J. Berzelius,1838

In 1828 Wohler was able to synthesize the first organic

compound in his laboratory .

P a g e 13
This discovery went against theory of that time
called vitalism which stated that organic matter is produced
only inside living organisms and cant be produced in labs .
For this reason berzelluis prposed that there is a sharp
boundary existed between organic and inorganic compounds
eg Urea was discovered in 1799 and could until then only be
obtained from biological sources such as urine.
However Whler reported to Berzelius after his discovery
"I must tell you that I can make urea without thereby needing
to have kidneys, or anyhow, an animal, be it human or dog".
It is argued that organic chemistry started with the synthesis
of oxalic acid in 1824 by Whler from the inorganic
precursor cyanogen . Oxalic acid is produced from
degradation of both Vitamin C and Glycine in Humans.
After Wohlers discovery many scientists abandoned vitalism
theory . It was not untill 1845when Kolbe converted carbon
disulphide (inorganic) to acetic acid (organic) that vitalism
started to lose supporters in great number . However Pasteur
never abandoned Vitalism untill he died . the theory Came to
an end in 1897 by the famous experiment of Eduard
Buchner.

P a g e 14
In 1833 Anselme payen and jane Francios(chemists at French
sugar Factory) extraced diastase enzyme from malt.
the name of enzyme is taken from a Greek word which means to
separate and refers to any enzyme catalyzes conversion of starch
into maltose, this enzyme is important for seed Germination
when a plant seed starts its Growth as it converts stored starch
into sugars so that seed can extract energy needed for its
Growth.

The only known form of isomerism at pasteur time was

structural isomerism in which two compounds have the same
number and type of elements but differ in their structure (eg
Glucose and Fructose) . they differ in both chemical and
physical characters . However , pasteur found compounds that
P a g e 15
have the same chemical properties and differ only in some
physical properties (deviation of polarized light in polarimeter.
Thus introduced a new type of isomerism in which 2 compounds
have the same structural formula but differ In direction of atoms
in the space (optical isomerism).

In 1843 Louis Pasteur encountered the phenomenon of optical

activity during his investigation of the crystalline sediment that
accumulated in wine casks (a form of tartaric acid called
paratartaric acidalso called racemic acid, from Latin racemus,
bunch of grapes).
He used fine forceps to separate two types of crystals identical
in shape but mirror images of each other. Both types proved to
have all the chemical properties of tartaric acid, but in solution
one type rotated polarized light to the left (levorotatory), the
other to the right (dextrorotatory).

Polarimeter

P a g e 16
1.sodium lamp(monochromatic light) 2.monochromatic light vibrates in all planes.
3.polarizer (prism). 4.monochromatic but of separate wave length.
5.tube containing optically active solution 6. note change in direction of light as it passes.
7.analyzer (prism which can be moved). 8. Eye of person who conduct the experiment.

The solution in container (5) changes direction of light passing through it.

D- Glucose (DDLD) and L Glucose (LLDL) are enantiomer of

each other(mirror images).

D Glucose and D- Talose are diastereoisomer of each other.

D-Glucose and D- Galactose are special type of

diastereosiomers (ie they are epimers).

P a g e 17
 optical isomer

diastereoisomer enantiomer

Epimer diastereoisomer

D refers to chemical structure of molecule (ie the OH attached

to the carbon next to the terminal carbon is directed to the right
in space) where d refers to the substance rotates polarized light
to the right in polarimeter (physical character). Not each D is d .
D-Glucose is (+112) so it is d (rotates polarized light 112
degree to the right ) whereas its enantiomer L- Glucose is
(-112) so it is l or levorotatory(rotates polarized light 112
degree to the left.
D-Fructose is (-92) l or levorotatory whereas L- Fructose is
(+92) d or dextrorotatory .

In 1857 Pasteur showed that exposing yeast to air (aerobic

condition) causes yeast cell growth to increase, however, in
anaerobic condition fermentation rate decreases its Growth.
The effect can be explained; as the yeast being facultative
anaerobes can produce energy using two different metabolic
pathways:
A) When oxygen concentration is low(anaerobic condition), the
product of glycolysis, (pyruvate), is turned into ethanol and
carbon dioxide(fermentation) this occurs when using yeast in
bakery where Carbon dioxide increases gas filled spaces inside
P a g e 18
Flour the energy production efficiency is low
(2 moles of ATP per mole of glucose)and so less growth. Under
anaerobic conditions, the rate of glucose metabolism is faster,
but the amount of ATP produced (as already mentioned) is
smaller.
In this way the sprinter at the start of a race when Oxygen
supply is deficient uses the anaerobic pathway execisvely to
provide the same amount of ATP as that provided by aerobic
pathway . it is sort of adjusting to the Environment(low oxygen
supply) to generate the needed ATP. This is the process used
in alcohol(ethanol ) production where yeast is kept in anaerobic
conditions to act on grapes (rich source of Glucose).
B)If the oxygen concentration is increased, pyruvate is
converted to acetyl CoA by pyruvate dehdrogenase complex
inside Mitochondria that can be used in the citric acid cycle,
which increases the efficiency to 36 moles of ATP per mole of
glucose and. so it is advantageous for yeast to undergo Krebs
Cycle in the presence of oxygen, as more ATP is produced from
less glucose this conserves Glucose for other yeast to replicate
themselves (available food). breeding yeast is done in aerobic
conditions.
When exposed to aerobic conditions, the ATP production
increases and the rate of glycolysis and fermentation slows
down, because the ATP produced and Citric acid act as
an allosteric inhibitor for phosphofructokinase 1, the key
regulatory enzyme in the glycolysis pathway.

P a g e 19
This mechanism avoid over Generation of ATP when not
needed. In humans it is said that Krebs cycle inhibits Glycolytic
pathway instead of fermentation in bacteria.
in 1860 Pasteur recognized that Enzymes were essential for
fermentation but he links their action to structure and life of
Yeast as whole being still convinced with Vitalism.

P a g e 21
we recognize that each tissue and, more generally, each
cell of the organism secretes . . . special products or
ferments into the blood which thereby influence all the
other cells thus integrated with each other by a
mechanism other than the nervous system.

Charles douard Brown-Squard

He was one of the first to announce the existence of substances\

secreted into the bloodstream to affect distant organs now
known as hormones.

In 1856 he demonstrated that removal of the adrenal glands

resulted in death, due to lack of essential hormones.

P a g e 21
At age 72, at a meeting of the Societie de Biologie in Paris,
Brown-Squard reported that hypodermic injection of a fluid
prepared from the testicles of guinea pigs and dogs leads to
prolonged human life. It was known, among scientists as the
Brown-Squard Elixir.

He was the first scientist to work out the physiology of the

spinal cord, demonstrating that the decussation of the fibres
carrying pain and temperature sensation occurs in the cord itself
near central canal and fibers form Lateral spinothalamic tracts
reach thalamus then cerebral cortex carrying pain and
Temperature sensation.

His name was immortalized in the history of medicine with the

description of a syndrome which bears his name (Brown-
Squard syndrome) due to the hemisection of the spinal cord,
which he described after observing accidental injury of the
spinal cord in farmers cutting sugar cane in Mauritius.

P a g e 22
in 1851, he was sent to the University of Vienna, at the
monasterys expense, to continue his studies in the sciences.
While there, Mendel studied mathematics and physics under
Christian Doppler, after whom the Doppler effect of wave
frequency is named; he studied botany under Franz Unger, who
had begun using a microscope in his studies, and who was a
proponent of a pre-Darwinian version of evolutionary theory.

In 1853, upon completing his studies at the University of

Vienna, Mendel returned to the monastery in Brno and was
given a teaching position at a secondary school, where he would
stay for more than a decade. It was during this time that he
began the experiments for which he is best known.

An Austrian monk living in a Monastery , Mendel was

conducting hybridization experiments in a garden of peas .
Between 1853and 1863 he cultivated and tested nearly 5000
pea plants From these experiments, he came out with two
generalizations which later became known as Mendelian
inheritance.

In 1865, Mendel delivered two lectures on his findings to the

P a g e 23
Natural Science Society in Brno, who published the results of
his studies in their journal the following year, under the title
Experiments on Plant Hybrids. Mendel did little to promote his
work. References to his work from that time period indicated
that much of his work had been misunderstood.

It was generally thought that Mendel had shown only what was
already commonly known at the time(that hybrids eventually
revert to their original form) this was the common belief in that
time and this is scientifically wrong and was not related to
results of Mendels experiments .

Furthermore, Mendel's findings were not viewed as being

generally applicable, even by Mendel himself, who dias that
they are only applied to certain species or types of traits. Of
course, his system eventually proved to be of general application
and is one of the foundational principles of biology.

The major block is that they are only applicable to specific

binary characteristcs like those studied by Mendel on peas (eg
green versus yellow color of pea seed ) however characteristics
which involved multible gene interactions cant be completely
understood by using Mendel Laws of inheritance which are so
simple .

In 1900, however, his work was rediscovered by 3 European

scientists.

P a g e 24
Mendel's Laws of Inheritance

Law of Segregation During gamete formation, the alleles for

each gene segregate from each other so that each gamete carries
only one allele for each gene.
Union of gametes results in Zygote formation which contains 2
alleles for each gene , of from fathers gamete and the other from
mothers gamete.

Law of Independent Assortment : states that alleles of different

genes assort independently of one another during gamete
formation.(in this example one gene for color , the other for
tail). Each gene is expressed by 2 alleles which are distributed to
gametes independent of alleles of the other gene ie free
distribution all possilities are possible).

P a g e 25
Law of Dominance : Some alleles are dominant while others are
recessive; an organism with at least one dominant allele will
display the effect of the dominant allele ie recessive alleles will
always be masked by dominant alleles. So the predominant
character can be genetically pure (Homozygos) or not pure
(Heterozygos) while the recessive character will always be pure.

In 1869 Miescher isolated phosphate rich chemicals from White

Blood Cells at University of Tubingen in Germany. Later,
Miescher raised the idea that the nucleic acids could be involved
in heredity.

P a g e 26
in his later years [Gibbs] was a tall, dignified gentleman, with a
healthy stride and ruddy complexion, performing his share of
household chores, approachable and kind (if unintelligible) to
students. Gibbs was highly esteemed by his friends, but
American science was too preoccupied with practical questions
to make much use of his profound theoretical work during his
lifetime. He lived out his quiet life at Yale, deeply admired by a
few able students but making no immediate impress on
American science commensurate with his genius.
J. G. Crowther,

It is one of the striking features of the work of Gibbs, noticed by

every student of thermodynamics and statistical mechanics, that

P a g e 27
his formulations of physical concepts were so felicitously
chosen that they have survived 100 years of turbulent
development in theoretical physics and mathematics.

A. S. Wightman

Gibbs received the first Ph.D. degree in engineering granted in

the US ias aa 1873 e lilpad es the following work.

Gibbs worked at a time when US community stressed on

applicalbe not theoritical science. His research was not easily
understandable to his students or his colleagues and he made no
effort to popularize his ideas or to simplify them to make them
more accessible.

His work related to Biochemistry :

the most useful criterion for predicting the spontaneity of a

process is the free energy which is given the symbol G .
However it is impossible to measure absolute values of
energy(G) so we use free energy change which can be measured
( G).

the value of free energy change gives information about the

spontaneity of a reaction .

free energy change = energy of products energy of reactants.

If G is negative this indicates that energy of product is less

than reactant so energy is released (exergonic reaction) and this
type of reactions is spontaneous.eg Aerobic metabolism of
glucose.
If G is positive this indicates that energy of product is
greater than energy of reactant so energy is absorbed for
reaction to proceed(endergonic reaction) this type of reactions is
not spontaneous and needs energy from outside the system to
proceed.eg phosphorylation of ADP to ATP.

P a g e 28
If G is zero this means that both energy of products and
reactants is equal and the reaction is reversible at equilibrium
state.

In this reversible reaction we can relate delta G to standard delta

G( which is measured for reaction under the standard conditions
a temperature of 273 K (0 C) and an absolute pressure of 0.987
atm).

G0 = standard delta G.
R is the Gas constant = 8.3 J mol-1 K-1
T= absolute temperature = 273 Kelvin.

When the reaction is at equilibrium G becomse zero as we

mentioned before so the above relation becomes as follows .

From this relation we can calculate standard delta G for specific

reaction by calculating Keq for this reaction . R is constant and
T =273 K.
G0 is specific for each reaction because each reaction has its
own equlibrium constant.

P a g e 29
The symbol G is given to free energy in the honor of Gibbs . his
work is the basis of biochemical thermodynamics and he is
considered by some to have been the greatest scientist born in
the United States.

The total energy of the universe is constant; the total

entropy is continually increasing.
Rudolf Clausius,

In 1875 the relation between the first 2 laws of thermodynamics

and G0 possible when he could measure change in entropy S.
If we considered life number of reactions occuring at cellular
level . then we can determine if Life is possible(spontaneous) in
terms of thermodynamics by calculating G0 of reactions
occuring at cellular level.

The first law of thermodynamics states that it is impossible to

convert energy from one form to another at greater efficiency
than 100%. This goes with law of conservation of energy. The
first law focuses on enthalpy which is the heat of a reaction at
constant pressure this is easy to measure.

The second law states that even 100% efficiency during energy
transfere is impossible . The second law focuses on Entropy

P a g e 31
which is harder to measure but Ludwig deduced an equation for
measuring it S = K log W .

These 2 laws of thermodynamics can be related to the free

energy change by means of a well-Known equation.this
equation was deduced by J.Willard Gibbs but he couldnt
measure S.

In fact entropy means randomness to simplify if you entered a

room of a teenager you find everything not present in its proper
place , there is chaoes every where , there is randomness and
entropy in this case is increased. if a reaction has positive
change in entropy so free energy change is negative value and
the reaction is spontaneous .
Example of spontaneous reaction is oxidation of Glucose inside
your body . you take only one molecule of Glucose from the
environment and convert it into 6 molecules of Carbon dioxide
and 6 molecules of Water which you breathe out this of course
increases the randomness in the environment around you
(instead of one Glucose there are now 12 compound floating
freely in the atmosphere and getting away from each other
(randomness ,disorder and chaoes ).

P a g e 31
7 molecules 12 molecules

Also entropy can occur at the level of energy : to make it simple

, we all know that hot steam can do work you can see that if
have a tea pot full of water boiling on the burner , the cover will
shake when water boils this means that steam does work on the
cover . imagine that you turned of he burner and let the teapot
lose heat to the air in the kitchen(ie. universe) .

P a g e 32
the heat now can not do work because it will act on air in the
kitchen increasing its randomness and will never spontaneously
go back from air to tea pot to increase its temperature again.

When we eat our meals we get energy released + building

blocks. The reactions which give energy as Glucose oxidation
release carbon dioxide and water which increase entropy in the
surrounding .

While the energy released part of it (40%) is used to get

building blocks (free Amino acids) linked together In special
way to form Protein which is very ordered and shows no
entropy .
so our body becomes more ordered (less entropy) but make
world less ordered(more entropy).

For life to occur (reactions at cells occur spontaneously) either

energy should be released from reactions or the products show
more entropy than reactants or both. This drives the reactions
forwards.

The first one to refer to entropy was Boltzmann . his equation

for entropy of the universe was one of his supreme
achievements . this equation is carved on his tombstone.

P a g e 33
Since the proteins participate in one way or another in all
chemical processes in the living organism, one may
expect highly significant information for biological
chemistry from the elucidation of their structure and their
transformations.
Emil Fischer

in 1875
he discovered phenylehydrazine This compound would play a
critical role in Fischer's later research on sugars.
he could distinguish different sugars based on their reaction of
phenylhydrazine with sugars. the reaction takes place in 2
steps and results in production of compounds easily
distinguished undermicroscope based on the characteristic
shape of their crystals. For an osazone to be formed a free
carbonyl group (aldehyde or ketone group should be present.

P a g e 34
in 1884
he discovered that adenine , xanthines , caffeine , uric acid and
Guanine all belonged to the same family of compounds which
he called purine.
In 1890
he also proposed a "Lock and Key Model" to visualize the
substrate and enzyme interaction. Though, later studies did not
support this model in all enzymatic reactions.

between 1891 and 1894

he established the stereochemical configuration of all the known
sugars and exactly foretold the possible isomers, by application
of the theory of the asymmetrical carbon atom. for example
aldohexoses have 16 possible stereoisomers because they
contain 4 assymetric carbon atoms.
His greatest success was his synthesis of glucose, fructose and
mannose in 1890, starting from glycerol.

Between 1899 and 1908

he studied proteins discovering a new type, the cyclic amino
acid proline and hydroxyproline. He was able to establish the
type of bond that would connect them together in chains named

P a g e 35
 the peptide bond. In 1901 he discovered, in collaboration with
Ernest Fourneau the synthesis of the dipeptide glycylglycine.
and in that year he also published his work on the hydrolysis of
Casein..
in 1904 he also introduced Barbiturates(a class of sedative drugs
used for insomnia, epilepsy, anxiety, and anesthesia ) together
with Joseph von Mering.

He won Noble Prize in 1902 for his great contributions.

Pulverize : to crash something

into powder.

In 1897 Buchner yielded another blow to the Vitalism theory

when he produced a cell free extract of Yeast(no living cells as
Pasteur argued) which could Ferment sugar .

The cell-free extract was produced by combining dry yeast cells

and quartz then pulverizing the yeast cells .

Microscopic investigation revealed no living yeast cells in the

extract This mixture would then become moist.

this moist mixture was added to Glucose , Fructose and

mannose and Carbon dioxide was seen to evolve .

Buchner hypothesized that yeast cells secrete proteins into their

environment in order to ferment sugars, but it was later found
that these proteins are secreted inside the cells themselves not in
P a g e 36
the environment .these proteins are known now as enzymes of
Glycolysis and Fermentation.
He received Noble prize in 1907 for this work.

Proteins : for Growth

and regeneration of
tissues .
Carbohydrates : as rapid
source of Energy.
Fats : energy stores..
Water :essential for life.
Minerals : osmotic
pressure and electic
activity.
Vitamins: Co-enzymes
annd Hormones(vit D)

In 1912 Hopkins published the work for which he is best known,

demonstrating in a series of animal feeding experiments that
diets consisting of pure proteins, carbohydrates, fats, minerals,
and water fail to support animal growth. This led him to suggest
the existence in normal diets of tiny quantities of unidentified
substances that are essential for animal growth and survival.
These hypothetical substances he called accessory food
factors, later renamed vitamins.They are essential for life since
most of them can not prepared in human body.

He later found that margarine nutritional value was less than

butter as it lacked Vitamins A and D .

He also discovered Glutathione and identified its tripeptide

structure. He won 1929 Nobel Prize in Medicine.
P a g e 37
 Leonor Michaelis Maud Leonora Menten

Nearly 100 years ago in 1913

Michaelis and Menten published their now classic paper
[Michaelis, L., and Menten, M. L. (1913) Die Kinetik der
Invertinwirkung. Biochem. Z. 49, 333369] in which they
showed that the rate of an enzyme-catalyzed reaction is
proportional to the concentration of the enzymesubstrate
Complex. They postulated that the enzyme first combines
reversibly with its substrate to form an enzyme-substrate
complex in a relatively fast reversible step:

The ES complex then breaks down in a slower second

step to yield the free enzyme and the reaction product P:

Because the slower second reaction must limit the rate of the
overall reaction, the overall rate must be proportional to the
concentration of ES.

P a g e 38
This equation calculates intial velocity from substrate
concentration . Note that Vmax and Km are constants.

Here, V max represents the maximum rate achieved by the

system, at maximum (saturating) substrate concentrations. The
Michaelis constant K m is the substrate concentration at which
the reaction rate V intial is half of Vmax .

The chromosomal theory of inheritance does not mean that

scientists believe Nucleic acids are the compounds involved in
Hereditary because chromosome= protein + nucleic acids . and
since they thought that proteins are more complex than
nucleic acids so they believed that proteins are resposible for
ccarrying genetic information which is ofcourse untrue

As a graduate he did his graduate work at Johns Hopkins in the

field of Zoology but turned to Genetics on rediscovery of
Mendle work in 1900.

in 1904 he was asked by his friend to Join Colombia University

and there he established his (Fly room) to determine how
species change over time .

Though initially being against the chromosomal theory of

inheritance , he became leading supporter of this idea showing

P a g e 39
that genes are found in series on chromosomes this brought the
theory to universal acceptance by 1915.

in 1933 he received Noble Prize for medicine for his work on

establishing the chromosomal theory of inheritance.

He invented the analytical ultracentrifuge in 1925.

Ultracentrifuge is often used to detect the rate at which a

molecule travels to the bottom of a test tube under the
centrifugal force of a centrifuge. The sedimentation coefficient
is the ratio of the speed of a substance in a centrifuge to its
acceleration in comparable units.

A substance with a sedimentation coefficient of 40S will travel

at 40 micrometers per second (=40106 metre/second) under
the influence of an acceleration of 107 metre/second2.
P a g e 41
Since sedmentation coefficient is a ratio : 40 . 10-6 metre /second
divided over 107 metre /second2 = 40 . 10-13 second
40 . 10-13 second = 40 Svedberg.
So one Svedberg unit equals 10 -13 second.

Centrifugal acceleration is calculated as following( r2)

where r is the radial distance from the rotation axis measured in
metres and is the angular velocity measured in radians per
second.

The sedimentation coefficient increases with the molecular

weight of the sedimenting Particle but is not directly
proportional to it because the particles shape also affects
sedimentation rate. we can make acceleration constant when we
compare different particles so when the Value of Sedimentation
cooefficient in Svedberg increases this means its velocity is
increased and so it has more weight or different shape from the
other particles or both.

The sedimentaion coefficient of Eukaryotic ribosome is 80S but

when dissociates it produces 40S and 60S particles .
40S + 60S 80S . so the values are not additive because there is
change in shape when ribosome dissociate however weight is
constant.

so sedimentation coefficient depends not only on weight but

also on shape(the shape of intact ribosome makes it harder to
sediment than its 2 units separately).

P a g e 41
The test tubes are put in their place and spinning at great
velocity creates great acceleration allows particles to sediment
and since we can adjust acceleration , the sedimentation velocity
will depend only on weigh and shape of particles

The unit of sedimentation coefficient is named after the

Swedish chemist Theodor Svedberg.

He won Noble Prize in 1926 for his invention and studying

colloids and proteins by this device.

P a g e 42
At Cornell , the American Scientist started his research in
attempt to isolate enzymes in pure form .

His work was unsuccessful at first and his colleagues were

doubtful believing that what he was trying to achieve was
impossible.

However in 1926 he isolated the first enzyme Urease In

crystallized form and showed that Urease enzyme was a protein
this was the first scientific proof that enzymes are proteins.

P a g e 43
He showed in 1928, that tumors have a higher rate of glucose
metabolism than other tissues.

He also purified and crystallized seven of the enzymes of

glycolysis (Note that the first purified enzyme was by James .B.
Summer).
He invented the Warburg manometer which measured directly
the consumption of oxygen by monitoring changes in gas
volume, and therefore allowed quantitative measurement of any
enzyme with oxidase activity.

He made Great contributions to many areas of biochemistry,

including respiration, photosynthesis, and the enzymology.
He trained at chemistry lap of Emil Fischer and Won Noble
Prize in 1931 in medicine.

P a g e 44
Formation of liver glycogen from lactic acid is thus seen
to establish an important connection between the
metabolism of the muscle and that of the liver. Muscle
glycogen becomes available as blood sugar through the
intervention of the liver, and blood sugar in turn is
converted into muscle glycogen. There exists therefore a
complete cycle of the glucose molecule in the body . . .
Epinephrine was found to accelerate this cycle in the
direction of muscle glycogen to liver glycogen . . . Insulin,
on the other hand, was found to accelerate the cycle in
the direction of blood glucose to muscle glycogen.

C. F. Cori and G. T. Cori

He and his wife defined Cori cycle in 1929.

P a g e 45
The cycle's importance is based on the prevention of lactic
acidosis in the muscle under anaerobic conditions(lactic acid
accumulate in anaerobic conditions because Krebs cycle is
blocked in absence of oxygen.

Normally before acidosis happens the lactic acid is moved out

of the musclet o the liver where it is converted to Glucose.

Since lactate is of no use in muscle under anaerobic condition ,

this cycle is important for conservation of Energy for the cell.

For these discoveries, they received the Nobel Prize in Medicine

in 1947. 6 of scientists of trained with the Cori became Noble
Laureates.

P a g e 46
He discovered deoxyribose in 1929 . Not only did Levene
identify the components of DNA,but he also showed that the
components were linked together in the order phosphate-sugar-
base to form units this is true for DNA structure. He called each
of these units a nucleotide

He also stated that the DNA molecule consisted of a string of

nucleotide units linked together through the phosphate groups,
which are the 'backbone' of the molecule.

He stated that the structure of DNA was only four nucleotides

per molecule. He even declared that it could not store the
genetic code because it was chemically far too simple.(these 2
pieces of information are not true and were scientifically proven
to be wrong after that)

However, his work was a key basis for the later work that
determined the structure of DNA. Levene published over 700
original papers and articles on biochemical structures. Levene
died in 1940, before the true significance of DNA became clear.

P a g e 47
 The first one to
discover vitamins
and refer to their
importance for
health
wasFrederick
Gowland Hopkins

L-Ascorbic acid
(vitamin C) is a
white, odorless,
crystalline powder.
It is freely soluble
in water and
relatively
insoluble in
organic solvents.
In a dry state,
away from light, it
is stable for a
considerable
length
of time.
. . . from this misfortune, together with the unhealthiness
of the country, where there never falls a drop of rain, we
were stricken with the camp-sickness, which was such
that the flesh of our limbs all shrivelled up, and the skin
of our legs became all blotched with black, mouldy
patches, like an old jack-boot, and proud flesh came
upon the gums of those of us who had the sickness,
and none escaped from this sickness save through the
jaws of death. The signal was this: when the nose began
to bleed, then death was at hand . . .
from The Memoirs of the Lord of Joinville, ca. 1300

This describes the what happened to Louis IXs army preceding

the battle of Fariskur, where the scurvyweakened the army and
this is one cause for their defeat by Egyptian army.

This describes scurvy which caused death of thousands of sailors

around the wordl and the cause was unknown. But now it is
known that sailors used preserved food for their long journeys
which did not contain fresh fruit or Vegetables so it lacked
Vitamin C which is needed for synthesis of collagen so sailors

P a g e 48
suffered from degradation of connective tissure rich in collagen as Blood vessels ,
gums of teeth , periodontal ligament ,

Humans and other animals (guinea pigs gorillas and fruit bats ) lack enzymes
needed for synthesis of ascor bic acid from Glucose.

In 1747, James Lind, a Scottish surgeon in the Royal Navy, carried out the first
controlled clinical study in recorded history.

During an extended voyage , Lind selected 12 sailors suffering from scurvy and
separated them into groups. All 12 received the same diet, except that each group
was given a different treatment . The sailors given lemons and oranges recovered
and returned to duty. The sailors given boiled apple juice improved slightly. The
remainder continued to deteriorate.

In 1930, Svirbely, working with Szent-Gyrgyi, conducted a

landmark experiment on guinea pigs, which, like humans, must
ingest vitamin C to maintain health since it can not synthesize it
from Glucose as do other animals like goats.

Svirbely divided the animals into two groups:

A)one that received boiled food (Heat destroys vitamin C)

B)the other that was fed food rich in synthetic ascorbic acid.

The latter group grew healthy, while the first group of guinea
pigs developed scurvy-like symptoms and then died.

Svirbely and Szent-Gyrgyi decided ascorbic acid was indeed

the long sought vitamin C.

In 1933, Szent-Gyrgyi wanted to find additional, natural

sources of ascorbic acid for further study.

Although orange juice and lemon juice have high levels of

ascorbic acid, they contain sugars that make purification
extremely difficult.

Szent-Gyrgyi solved the problem by paprika. Szeged in

Hungary is the paprika capital of the world, where salt and
paprika shakers are found on every restaurant table.
P a g e 49
Szent Gyorgi wrote in his autobiography (one night , my wife
served me fresh red paprika for supper . I did not feel like eating
it so I thought of a way out. Suddenly it occurred to me that this
is the one plant I had never tested. I took it to the laboratory ...
[and by] about midnight I knew that it was a treasure full of
vitamin C.)

Within several weeks Szent-Gyrgyi had produced three pounds

of pure crystalline ascorbic acid, enough to show- when fed to
the vitamin C-deficient guinea pigs -that the acid was equivalent
to vitamin C.

Szent-Gyrgyi went on to identify and study actin and myosin,

proteins responsible for muscle contraction, and demonstrated
that the compound adenosine triphosphate (ATP) is the
immediate source of energy necessary for muscle contraction.

Szent-Gyrgyi then turned to the study of organic compounds

known to play a part in the breakdown of carbohydrates to
carbon dioxide, water.

His work laid the foundation for Sir Hans .

He received the Nobel Prize in 1937.

P a g e 51
I chose the study of the synthesis of urea in the liver because it
appeared to be a relatively simple problem.
Hans Krebs

He discovered urea cycle . This cycle was the first metabolic

cycle discovered five years before the discovery of the Krebs
cycle by Hans Krebs and Kurt Henseleit.

If citrate is added the rate of respiration is often increased . . .

the extra oxygen uptake is by far greater than can be accounted
for by the complete oxidation of citrate . . . Since citric acid
reacts catalytically in the tissue it is probable that it is removed
by a primary reaction but regenerated by a subsequent
reaction.
H. A. Krebs and W. A. Johnson, article in Enzymologia, 1937

The German-born Jew who trained in medicine and chemistry,

was forced out of his position at the University of Freiburg as
the Nazis rose to power in the early 1930s.ma a depac e eh
Ga he d ae n oe c ama a he joined the faculty of the
University of Sheffield in 1935.

P a g e 51
In March 1937, Krebs and a colleague cut the breast of a
freshly killed pigeon in their lab, suspended it in solution, and
observed its metabolic rate decline over the next half hour.
By adding a salt of citric acid, however, they were able to keep
ama ai ua eoiva ee am aa aiha more than normal(ie without
citrate).

Additional experiments revealed the cyclical nature of the

pathway, which regenerates citric acid with each cycle and
releases ATPama daoo p ihe d a a d du a dd.

Krebs submitted his findings to Nature(scientific Journal)ama a

ama ieu eo aeu ac ae pupoi m e al e aidoa .

Tmi ae ama ei a aiha i hd de aa , eeaa mevi puloi mac

more than fifty papers, that I experienced a rejection or semi-
ajadaie ,
Hans Krebs

He resubmitted his findings to the journal Enzymologia in

Holland, where they were published within 2 months.

I 1988, 7 dae eeaa e al caeam, e aciae puloi mac e oaaaa

in Nature saying the rejection ee e al e aidoe is ama jeu eo
he a mehaeuo a e .

P a g e 52
This was the response of the Journal Nature refusing to puplish
for Krebs.

Several components and reactions of the citric acid cycle were

established in the 1930s by the research of the Nobel laureate
Albert szent for his discoveries pertaining to Fumaric acid a key
component of the cycle .

The citric acid cycle itself was finally identified in 1937 by

Hans Krebs for which he received the Nobel Prize for
Physiology or Medicine in 1953.

P a g e 53
 Outlive : means to live
longer than sth else

This is because the

protein theory of
genetic information
was proven to be
untrue because DNA
The one gene-one enzyme hypothesis is is the carrier of
the idea each gene is responsible for genetic information ,
producing a single enzyme that in turn However one gene
one enzyme theory is
affects a single step in a metabolic still true because each
pathway . gene in DNA codes for
specific enzyme that is
The concept was proposed by George resposible for a step
Beadle and Edward Tatum in an in the metabolic
influential 1941 paper. pathway.

By 1945, Beadle, Tatum and others, working with Neurospora

and other model organisms such as E-coli had produced
considerable experimental evidence that each step in a metabolic
pathway is controlled by a single gene.

In a 1945 review, Beadle suggested that "the gene can be

visualized as directing the final configuration of a protein
molecule and thus determining its specificity."

He also argued that "for reasons of economy in the evolutionary

process, one might expect that with few exceptions the final
specificity of a particular enzyme would be imposed by only one
gene."

At that time, genes were widely thought to consist of proteins

and nucleoproteins However, the proposed connection between
a single gene and a single protein enzyme outlived the protein

P a g e 54
theory of gene structure. In a 1948 paper, Norman Horowitz
named the concept the "one gene-one enzyme hypothesis".

This was the first

blow yielded to
Their experiment reported in 1944 that DNA is the
substance that induced bacterial transformation in an protein theory of
era when it had been widely believed that it was genetic information.
protein that served the function of carrying genetic
The second was by
information.
Chargaff (see later)
The third blow was
yielded by Hershey
and Chase (see
later).
Virulent : causing
damage and death.
Strain : means
specific type of
bacteria.

P a g e 55
At this experiment : rough strain of specific type
of bacteria is non virulent and causes no death of
mice while smooth type of the same bacteria is
virulent ,synthesize a poison and causes death of
the mice .

Heat killed smooth straim doesnt cause death of

mice because it is dead and cannot produce
poison.

Heat killed smooth strain + rough strain cause

death of mice . This means that genetic
information for production of the poison has been
transferred from Heat killed smooth type to the
living non virulent rough type. The rough type
now can express the genetic information
transferred to it and produce poison that kills
mice.

If we could purify the substance that has transferred and detect

its chemical nature , we can state if protein or DNA carries
genetic information . the substance that caused bacterial
transformation was proved to be DNA by the following :

The purification procedure Avery undertook consisted of first

killing the bacteria with heat and extracting the saline soluble
Components.

Next, the protein was periciptated using Chloroform and the

polysaccharide capsules of bacteria were hydrolyzed by an
enzyme An immunological precipitation caused by type-specific
antibodies was used to verify the complete destruction of the
capsules.

P a g e 56
Then, the active portion that caused bacterial transformation was
precipitated out by alcohol fractionation resulting in fibrous
strands that could be removed with a stirring rod.

Chemical analysis showed that the proportions of carbon,

hydrogen, nitrogen, and phosphorus in this active portion were
consistent with the chemical composition of DNA. To show that
it was DNA rather than some small amount of RNA , protein or
some other cell component that was responsible for
transformation.

Avery and his colleagues used a number of biochemical tests.

They found that trypsin , chymotrypsin(cause protein
degradation) and ribonuclease(cause RNA degradation) did not
convert the active material to inactive one so that proteins and
RNA dont carry genetic information.

However treating the active substance by

"deoxyribonucleodepolymerase, that could break down DNA
destroyed the active substance and converted it to inactive
material that is not able to cause bacterial transformation.

P a g e 57
the discovery of the long-lived isotope of carbon,
carbon-14 by Samuel Ruben and Martin Kamen in 1940
provided the ideal tool for the tracing of the route along
which carbon dioxide travels on its way to carbohydrate.

Melvin Calvin

The calvin cycle was discovered by Melvin Calvin, James

Bassham, and Andrew Benson in 1950 at the University of
California, Berkeley by using the radioactive isotope carbon-14.

Calvin cycle is one of the light-independent reactions in

photosynthesis used for carbon fixation.One experiment after
another. By using radioactive substances such as radioactive
carbon in carbon dioxide, and stopping the process after known

P a g e 58
time intervals, and finding what substances were radioactive, the
full cycle was worked out.

Glucose is not a direct product of the Calvin cycle .the triose

Glyceraldhyde 3 phosphate is the direct product.

epyhelips yse3l sdl ipe ad hsahelpes ps ra syslsiy ihepsae

l sdl ipe ey l sdl splasde adshelide.

Glyceraldhyde 3 phosphate + Dihydroxy acetone phosphate =

Fructose 1.6 bisphosphate by Aldolase enzyme .

Fructose 1.6 bisphosphate to Fructose 6 phosphate then Glucose

6 phosphate then Glucose eventually is produced.

The Noble Prize in chemistry 1961 was awarded to Melvin

Calvin.

Photosynthesis consists of :
Light dependent reactions : provides energy in the form of ATP and
reducing power in the form of NADPH.

Light independent reactions : represent Calvin cycle in which

carbon dioxide is reduced using NADPH in the presence of ATP .

Melvin Calvin stressed on that the direct product is Glyceraldhyde 3

phosphate not Gluose.

P a g e 59
GAP is Glyceraldhyde 3 phosphate.

P a g e 61
He is best known for 2 rules regarding DNA structure :

The first and best known achievement was to show that in

natural DNA the number of Guanine units equals the number of
Cytosine units and the number of Adenine units equals the
number of Thymine units .

In human DNA A=30.9% and T=29.4%; G=19.9% and

C=19.8% . this disapproves Phoebus Levene hypothesis. Also
he met Watson and Crick to show them his discoveries which
later helped them to deduce the double Helix of DNA structure.

The second of Chargaff's rules is that the composition of DNA

varies from one species to another, in particular in the relative
amounts of A, G, T, and C bases. This made DNA a more
trusted candidate for the genetic information than protein.

These findings were published in 1950.

This is the second prove

that DNA carries genetic
information

P a g e 61
I believe that as the methods of structural chemistry are
further applied to physiological problems, it will be found
that the significance of the hydrogen bond for physiology
is greater than that of any other single structural feature.

Linus Pauling

Of all the natural systems, living matter is the one which,

in the face of great transformations, preserves inscribed in
its organization the largest amount of its own past history.

Emile Zuckerkandl and Linus Pauling

In 1951 he proposed the a-helix and the Beta-pleated sheet

structures for proteins.

P a g e 62
One person, Linus Pauling, has won two undivided Nobel
Prizes. In 1954 he won the Prize for Chemistry. Eight years later
he was awarded the Peace Prize for his opposition to weapons of
mass destruction.

Also in 1951 Dr. Linus Pauling and his colleague Dr. Harvey
Itano, discovered that the red, oxygen-carrying protein called
hemoglobin had a different chemical structure in persons with
sickle cell disease. This led Dr. Pauling to use the term
molecular disease for disorders that resulted from proteins
with abnormal chemical structures. Today, thousands of such
diseases are known but in 1951, sickle cell disease was the first.

This was also the first demonstration that Mendelian inheritance

determined the specific physical properties of proteins, not

P a g e 63
simply their presence or absence .It is the dawn of molecular
genetics.

Normal people and people with sickle cell

disease both have Hemoglobin in their RBCs
but differs in its structure . this means that
genetics not only determine the presence or
absence of a protein but also tne structure of a
protein

Alfred Hershey and Martha Chase

The HersheyChase experiments were a series of experiments

conducted in 1952 by Heshey and chase that helped to confirm
that DNA is the Genetic material .

Hershey shared the 1969 Noble prize for physiology and

medicine with max Delbruck and Salvador Luria for their
discoveries concerning the genetic structure of Viruses.

A bacteriophage infects bacteria . the bacteriophage has sulphur

labeled protein(red) and phosphorus labeled DNA(Green) . the
phage inserts its genetic material into bacteria then replicates
inside bacteria .then finally is released out of bacteria.
It was observed that labeled phosphorus (DNA)completely
enters bacteria while labeled sulfur(protein) remains outside. So
it was deduced that genetic material is DNA and not protein .

P a g e 64
 Watson and Crick

In an article that is dated 25 April 1953 and described as a pearl

of science Watson and Crick described their discovery of
Double helical structure of DNA.
Watson stated among other things that he and Crick had access
to some of Franklin's data from a source that she was not aware
of, and also that he had seenwithout her permissionthe B-

P a g e 65
DNA X-ray diffraction pattern obtained by Franklin and Gosling
in May 1952 at King's in London.

Photo 51 x-ray diffraction of wet DNA

showing the B form double helix

Crick plays a role in understanding the gemetic code :

It was clear that there must be some correspondence between the

linear sequences of nucleotides in DNA molecules to the linear
sequences of amino acids in proteins Crick played important
role in understanding the genetic code.

It is dictionary, which corresponds the nucleotide sequence of

mRNA and the amino acid sequence on the polypeptide chain of
protein.
Each individual word in the code is composed of three nucleotide
bases. The codons are presented in the messenger RNA.
The four nitrogenous bases are used to produce the three-base
codons. There are therefore 64 different codons, taken three at a
time.
The nucleotide sequences are always written from the 5'-end to the
3'-end.
Sixty-one codons code for the twenty amino acids in nature and
three codons code for the termination of the polypeptide chain
during protein biosynthesis

P a g e 66
 Characters of genetic code:
A.Specificity: (unambiguous): The genetic code is specific: A single
codon always codes for the same amino acid.
B.Universality: the codons are the same for amino acids in all species;
human, rabbit and fish.
C.Redundancy (degenerate). A single amino acid may have more than
one codon For example; serine is specified by six different codons. If a
given amino acid has more than one codon the first two bases will be the
same, but the third base is different.
D.Genetic code is non-overlapping and commaless: That is the codon
is read from a fixed starting point as a continuous sequence of bases,
taken three at a time, for example ABCDEFGHUKL is read as
ABC/DEF/GHI/JKL/ without any punctuation between the codons.

Watson directed the human genome project at the national institutes of

Health .

P a g e 67
Till that time proteins were considered amorphous substances
but sanger proved that they have definite chemical structure and
determined sequence of aminoacids that is characteristic for
each protein , this helped very much in knowing more about
genetic code.

In 1953 sagner used the Sanger Reagent" fluorodinitrobenzene"

to react with the exposed amino groups in the insulin and in
particular with the N-terminal amino group at one end of the
polypeptide chain.

He then partially hydrolysed the insulin into short peptides,

either with hydrochloric acid or using an enzymes

The peptide containing the N-terminus could be recognised by

the yellow colour caused by the fluorodinitrobenzene

the identity of the labelled amino acid at the end of the peptide
was determined by complete acid hydrolysis and discovering
whichamino acid was attached to fluorodinitrobenzene.

he received a Noble Prize In chemistry for this work.

P a g e 68
When I first entered the study of hormone action, some
25 years ago, there was a widespread feeling among
biologists that hormone action could not be studied
meaningfully in the absence of organized cell structure.
However, as I reflected on the history of biochemistry, it
seemed to me there was a real possibility that hormones
might act at the molecular level.
Earl W. Sutherland, Nobel Address, 1971

P a g e 69
 In 1958 Sutherland made the discovery that would lead to his
1971 Nobel Prize in Physiology or Medicine for his discoveries
concerning the mechanisms of the action of hormones. It was
at that time that Sutherland isolated a previously unknown
compound, called cyclic adenine monophosphate (cAMP) and
proved that it had an intermediary role in many hormonal
functions. He trained with the Cori.

The DNA of the cells

grown in 15N medium
had a higher density
than cells grown in
normal 14N medium .

There have been 3 hypothesis for replication of DNA . Scientists

didnt know which one was true untill the experiment by
Matthew Meselson and Franklin Stahl.
In the semiconserative the
Conservative replication in In thedispersive model of parental double helix is
which DNA strands dont DNA replication, the parent separated into 2 strands (not
separate from each other and DNA strands are cleaved into conserved as entity) , each
new identical DNA double pieces of random size, then individual strand remains
strand is formed. joined with pieces of newly intact ( conserved) and acts
replicated DNA to yield as a Template for synthesis
daughter duplexes. of Complementary strand.

P a g e 71
If E. coli were grown for several generations in a medium with
15
N. their DNA is extracted from them and centrifuged on a salt
solution, the DNA separates out at the point at which its density
equals that of the salt solution.while if it is grown in a medium
with 14N , its DNA separates out also at at the point at which its
density equals that of the salt solution . and this will be less than
15
N.
The experiment (1958)

E. Coli were grown for several generations in a medium with

15
N so that their DNA contained 15N.

After that, E. coli cells with only 15N in their DNA were
transferred to a 14N medium and were allowed to divide; the
progress of cell division was monitored by microscopic cell
count untill cell population has just doubled.

DNA was extracted periodically and was compared to pure 14N

DNA and 15N DNA.

After one replication, the DNA was found to have intermediate

density between 15N and 14N .

Since conservative replication would result in equal amounts of

DNA of the higher and lower densities (but no DNA of an
intermediate density), conservative replication was excluded.

However, this result was consistent with both semiconservative

and dispersive replication. Semiconservative replication would
result in double-stranded DNA with one strand of 15N DNA, and
one of 14N DNA, while dispersive replication would result in
double-stranded DNA with both strands having mixtures of 15N
and 14N DNA, either of which would have appeared as DNA of
an intermediate density.

The authors continued to sample cells as replication continued.

DNA from cells after two replications had been completed was
found to consist of equal amounts of DNA with two different

P a g e 71
densities, one with the intermediate density, the other
corresponding to DNA from cells grown exclusively in 14N
medium.

This was inconsistent with dispersive replication, which would

have resulted in a single density, lower than the intermediate
density of the one-generation cells, but still higher than cells
grown only in 14N DNA medium, as the original 15N DNA
would have been split evenly among all DNA strands.

The Result was that DNA replication is semiconservative.

Red strand = DNA with N14

Blue strand = DNA with N15

P a g e 72
 John Kendrew and Max Perutz

Perhaps the more remarkable features of [myoglobin] are

its complexity and its lack of symmetry. The arrangement
seems to be almost totally lacking in the kind of regularities
which one instinctively anticipates, and it is more
complicated than has been predicted by any theory of protein
structure.
John Kendrew

The previous theories of protein structure predicted primary structure

(type and sequence of Amino acids) . the secondary structure of proteins
was proposed by Linus Pauling and Robert Corey(-helix or The -
sheet) . the tertiary structure discovered by John Kendrew Refers to
the manner in which the polypeptide chain (-helix) is bent or
folded to form a compact tightly folded structure forming
globular protein e.g M yoglobin.

In 1947 the MRC agreed to make a research unit for the Study
of the Molecular Structure of Biological Systems.

P a g e 73
The original studies were on the structure of sheep hemoglobin
but when this work had progressed as far as was possible using
the resources available.

Kendrew started the study of myoglobin , a molecule only a

quarter the size of the hemoglobin molecule.

His initial source of raw material was Horse Heart t, but the
crystals thus obtained were too small for X-ray analysis.

Kendrew realized that the oxygen-conserving tissue of sea

mammals could offer a better source of large myoglobin , and
the chance allowed him to studty Whale meat from Peru.

Whale myoglobin gave large crystals with clean X-ray

diffraction patterns.

However, the problem still remained, until 1953 Max Perutz

discovered that the problem in analysis of the diffraction
patterns could be solved by comparison of multiple patterns of
X-Ray diffraction from several crystals; one from the native
protein, and others that had been soaked in solutions of heavy
metals and had metal ions introduced in different well-defined
positions.

An electron density map at 6 angstrom resolution was obtained

by 1957.

By 1959 an atomic model could be built at 2 angstrom

resolution the 2 scientists shared 1962 Noble prize in chemistry.

P a g e 74
He was working at University of Aarhus as associate proffessor
in department of Biochemistry when he discovered the first Ion
transporter (sodium potassium pump) in 1959.

This transporter has many functions in humans :

It keeps resting membrane potential constant as it pumps 3

sodium Ions from inside the cell to the outside against
concentration gradient for 2 potassium ions pumped from
outside to inside the cell aslo against concentration gradient thus
requiring ATP molecule to be hydrolysed releasing energy
needed for transport.

It provides sodium gradient across cells to make secondary

active transport possible . this occurs for example in Gut for
absorption and in Kidney for reabsorption of Glucose ,
Aminoacids and other useful nutrients.
Since sodium potassium pump , pumps sodium outside the cell ,
there is increased sodium gradient across cell membrane. This
gradient allows Glucose or Amino acids to move along with Na+
across cell membrane by a mechanism known as secondary active
transport.

P a g e 75
When cells begin to swell this activates the pump to get some
ions out of the cells reducing osmolarity inside the cell and
preventing more water to enter cell by osmolarity which may
end in lysis and death of cell. It is to be noted that old RBCs
have less pumps so they are more liable to swell and lyse in
osmotic fragillity test than recent RBCs.

Osmotic fragillity test : RBCs are placed in solutions with

different osmotic pressures . ions tend to diffuse inside
the cell by concetration gradient then water follows
causing ballowning of cells and may cause lysis but the
pump , pumps them out to prevent lysis.

It is thought now that they are not only homeostatic but help as
signal transducer eg for ouabain.

Also have active role in function of purkinjie cell of cerebellum.

In 1997 he recieved one half of Noble Prize in chemistry for

this discovery.

P a g e 76
 Electrophoresis : separation of charged biomolecule depending on different
rates at which they migrae toward positive or negative poles when electric
field is applied.
Mobility through electric field in PAGE : is determined by
shape(confiurguration of molecule) , Mass of molecule and charge to mass
ratio . but when SDS is added to protein , the migration depends only on
mass because SDS denatures protein (turns protein to unstructured linear
chain) also keeps charge to mass ratio conistant with any protein , so
migration will depend only on mass , the increase in mass decreases
migration rate.

Polyacrylamide Gel electrophoresis was developed in 1959.

It possesses several electrophoretically desirable features that : It

is a synthetic, thermo-stable, transparent, strong, chemically
relatively inert gel, and can be prepared with a wide range of
average pore sizes.

Polyacrylamide gel electrophoresis (PAGE), describes a

technique widely used in analytical biochemistry to separate
macromolecules(eg proteins and DNA)

As with all forms of gel electrophoresis molecules may be run

in their native state preserving the molecules' higher-order
structure .

SDS(chemical denaturant) may be added : SDS binds to protein

at a ratio of one molecule of SDS per 2 peptide bonds ,causing
the polypeptide to unfold or denature . Also SDS separates
polypeptide compnents of multimeric protein as Hemoglobin
(separated to 4 polypeptides) . the total number of SDS each
bearing -1 charge is much greater than charges caused by
Aminoacids side chains in the polypeptide. Since the charge to
mass ratio of SDS-polypeptide complex would be
approximately equal in different polypeptides . so the physical
P a g e 77
resistance each polypeptide encounters during migration through
PAGE determines the rate of migration . Since larger
polypeptide would encounter greater resistance , polypeptide
migrate depending on their Molecular mass.

P a g e 78
RNA polymerase was discovered in 1960 by the 3 scientists this
enzyme catalyzes transcription of Genes on DNA it was first
separated from E.coli .

In Eukaryotes like humans there are 3 types of it one for m-

RNA another for t-RNA and the last one for r-RNA .

DNA-dependent RNA polymerase requires, in addition to a

DNA template, all four ribonucleoside 5_-triphosphates (ATP,
GTP, UTP, and CTP) as precursors of the nucleotide units of
RNA, as well as Mg+2. The protein also binds one Zn +2.The
chemistry and mechanism of RNA synthesis closely resemble
those used by DNA polymerases . RNA polymerase elongates
an RNA strand by adding ribonucleotide units to the 3hydroxyl
end, building RNA in the 5 3 direction.
In 1959 , Noble prize was awarded for Severo ochoa who
thought that he discovered RNA Polymerase but in fact it was
Polynucleotide phosphorylase.

P a g e 79
The fundamental problem of chemical physiology and of
embryology is to understand why tissue cells do not all
express, all the time, all the potentialities inherent in their
genome.
Franois Jacob and Jacques Monod

Today, the operon is simply defined as a cluster of genes

transcribed into a single mRNA molecule.

P a g e 81
Nevertheless, the development of the concept is considered a
landmark event in the history of molecular biology. The first
operon to be described was the lac operon in E-Coli. In lac
operon The 3 genes are transcribed into one large polycistronic
m-RNA molecule that contains multible idependent translation
start (AUG) and a stop codon for each of 3 genes . thus each
proteins is translated separately.

In 1961 the general theory of the operon was developed. This

theory suggested that in all cases, genes within an operon are
negatively controlled by a repressor acting on operator before
the first geneLater, it was discovered that genes could be
positively regulated and also regulated at steps that follow
transcription initiation. Therefore, it is not possible to talk of a
general regulatory mechanism, because different operons have
different mechanisms..

O = operator P = promoter I = laci(repressor gene).

1.In bacteria , the structural genes that code form the enzymes of
a metabolic pathway are often found grouped together on the
chromosome with regulatory genes that control transcription .
(genes of metabolic pathway + regulatory genes = operons).
Example is lactose operons in E-coli : this operons codes for 3
enzymes involved in the catabolism of lactose sugar . lac Z
codes for beta Galactosidase which hydrolizes lactose to
Glucose and Galactose , lac Y codes for permease which
facilitates the movement of Galactose into the cell , lac A gene
codes for thiogalactoside transacetylase whose function is not
known.these enzyme are expressed when there is lack of
P a g e 81
Glucose and lactose is the only sugar present in environemt . the
regulatory portion of this operons consists of 3 sites and 1
repressor gene . the first site is CAP binding site(positive
regulatory site) , the second is promoter site , the third is
operator site(negative regulatory site) . the repressor gene lacl
codes for a repressor protein .

3. when Glucose is the only sugar available : the repressor gene

produces repressor protein which binds to the operator site
which is upstream of promoter site and this blocks progress of
RNA polymerase so no transcription from genes of metabolic
pathway (structural genes). Because Glucose is available adenyl
cyclase is inhibited so no cAMP is available to combine with
repressor protein and form CAP.

4.when lactose is only available : small amount of lactose is

converted into allo lactose that binds to repressor protein and
changes its conformation so it cannot bind to operator site .
because no Glucose is available adenyl cyclase is available and
produces cAMP which also binds to repressor protein to form
CAP which binds to CAP binding site. This allows RNA
polymerase to effectively initiate transcription .

5. when both Glucose and lactose are available : no CAP is

formed so polymerase cannot effectively initiate transcription ,
although repressor protein is not bound to operator site . so three
genes are not expressed .
6. According to previous information : activation of this operons
occurs when there is not repressor protein bound to operator site
and there is CAP bound to CAP binding site .

Glucose is the main and important source of nutrition for E-

Coli.

The Noble prize in physiology was awarded for this work in

1965.

P a g e 82
In biochemistry allosteric regulation is the regulation of an
enzyme by binding an effector molecule at the proteins
allosteric site.leadign to
conformational change or other change in the effectiveness of
the enzyme. Positive allosteric regulator increases catalysis by
the enzyme while Negative allosteric regulator decreases
catalysis.for example the key enzyme of Glycolysis
(phosphofructokinase 1 is allosterically activated by binding
AMP and inhibited by binding Citrate or ATP).

Allosteric : means the other

site . ie. Site other than active
site

I concreted model :
enzyme has 2 conformations (Relaxed and Taut).

Relaxed conformation (R ) : this conformation binds substrate

tightly (catalytic site is complementary to substrate) .

Taut confroation (T) : which binds substrate less tightly.

Both conformations are in equilibrium with each other.

If the enzyme has 4 subunits : in presence of Substrate in high
quantity or activator this shifts equilibrium toward R state(4
subunits are conformed into R at the same time) .
P a g e 83
Also in the presence of inhibitor this shifts equilibrium to the T
state(inactive) .ie inhibitors can bind to the T state and stabilize
it whereas activator binds to R state and stabilize it .

II-sequential model : binding of substrate or activator in one

subunit induces conformational changes in the Catalytic site of
the same subunit then these changes are transferred to the
other subunit to become in R state. In this model inhibitor binds
to the T state and changes shape of Catalytic site so that it has a
lower affinity for binding substrate.

S= substrate . red circle = Relaxed . Green square = taut.

P a g e 84
 David Chilton Philips

The antibacterial property of hen egg white was first observed

by laschtschenko in 1909 however it was Fleming who gave the
name Lysozyme .
Lysozyme means enzyme that
causes lysis of bacteria.

Felming observed antibacterial action of this enzyme when he

treated bacterial cultures with nasal discharge of people with
head cold (the nasal discharge contained Lysozyme).

The 3D structure of the enzyme was first described by David

Philips in 1965 by using X-ray crystallography.

It was the 2nd protein structure and 1st enzyme structure revealed
by X-ray diffraction method. Also it was the first enzyme to be
fully sequenced that contains all twenty common amino acids.

P a g e 85
The first protein structure was myoglobin by x-ray diffraction method. See John Kendrew
before.

Crystallography is the science that examines the special arrangement of molecules in

solids. normally proteins and DNA are in soluble form in our body but in vitro when their
concentration is raised gradually they form Crystalls with special pattern ,then using X-ray
diffraction techniques ,photos are available to predict the 3D structure of protein /DNA
after several complex mathematical methods.

The RNA of the cell is partly in the nucleus,

partly in
particles i ama ddaepoe h e c pe aod e ama eouloa RNA
of the cell sap; many workers have shown that all these
three fractions turn over differently. It is very important to
realize in any discussion of the role of RNA in the cell
that it is very inhomogeneous metabolically, and probably
of more than one type.

Francis H. C. Crick

P a g e 86
The existence of t-RNA was first hypothesized by Francis Crick
on the assumption that there must exist an adapter molecule
capable of mediating the translation of the RNA alphabet into
the protein alphabet.this goes with Cricks role in identifying
genetic code which requires a molecule which translate codons
into Amino acids.

In 1965 Robert Holley at Cornell University reported the

primary structure and suggested three secondary structures.

The Cover leaf structure was then confirmed by X-ray

Crystallography.

P a g e 87
Supercoiling, in fact, does more for DNA than act as an
executive enhancer; it keeps the unruly, spreading DNA
inside the cramped confines that the cell has provided
for it.
Nicholas Cozzarelli

DNA supercoiling refers to the over- or under-winding of a

DNA strand.

Supercoiling is important in a number of biological processes,

such as compacting DNA to be easliy stored in the small sized
nucleus of cell . positive supercoils decrease transcription while
negative supercoils which means DNA double helix is relaxed
increase the rate of transcription also fascilitate progression of
replication of DNA as DNA polymerase wont progress untill
DNA is relaxed.

Additionally, certain enzymes such as topoisomerases are able

to change DNA topology to facilitate functions such as DNA
replication or transcription ie DNA gyrase can generate negative
supercoils into relaxed DNA and relax positively supercoiled
DNA. In contrast, topoisomerase I can relax negatively
supercoiled DNA but not positively supercoiled DNA

Mathematical expressions are used to describe supercoiling by

comparing different coiled states to relaxed B-form DNA.
many circular DNA molecules remain highly supercoiled even after they are extracted and
purified, freed from protein and other cellular components. This indicates that supercoiling is an
intrinsic property of DNA tertiary structure. It occurs in all cellular DNAs and is highly
regulated by each cell.

a branch of mathematics called topology, the study of the properties of an object that do not
change under continuous deformations. For DNA, continuous deformations include
conformational c hanges due to thermal motion or an interaction with proteins or other
molecules; discontinuous deformations involve DNA strand breakage. For circular DNA
molecules, a topological property is one that is unaffected by deformations of the DNA strands
as long as no breaks are introduced. Topological properties are changed only by breakage and
rejoining of the backbone of one or both DNA strands.

P a g e 88
 It was found that a bacteriophage that can grow well in one
strain ofEscherichia coli, for example E. coli C, when grown in
another strain, for example E. coli K, its yields can drop
significantly, by as much as 3-5 orders of magnitude.

The E. coli K host cell, known as the restricting host, appears to

have the ability to reduce the biological activity of the phage .

P a g e 89
 This ability to restrict replication of phage is
due to production of restriction enzyme which
breaks down phage DNA and cannt attack
bacterial DNA because it is methylated at
restriction site.

In 1970, Hamilton O Smith , Thomas Kelly and Kent

WelCox isolated and characterized the first type II restriction
enzyme from bacterium Haemophilus Influenzae This type of
restriction enzymes is more useful for laboratory use as they
cleave DNA at the site of their recognition sequence.

In 1978 Hamilton O Smith was awarded the Noble prize for

medicine as his work led to development of recombinant DNA
technology that allowed the large production of Human insulin
using E.Coli bacteria.

P a g e 91
Reverse transcriptase was discovered by Howard Temin and
isolated by David Baltimore in 1970 . this enzyme is used by
some Viruses to convert their RNA to double stranded DNA
which is able to recombine with Host Genome and replicates
itself like HIV virus which causes AIDS.

This enzyme first transcribes DNA from RNA which is opposite

to central Dogma of molecular biology which stated that RNA
are transcriped from DNA so the discovery was unpopular at
first .

After that the enzyme uses the transcriped single stranded DNA
to make a complementary strand resulting in double sttanded
DNA.

The 2 scientists shared Noble Prize for Medicine in 1975.

P a g e 91
The idea of recobinant DNA was first proposed by Peter Lobon
a graduate of Stanford University ,however it was not until 1973
when Stanely Cohen and Herbert Boyer published the first paper
about this technology the first drug generated from E.Coli using
this technology is human insulin.

In this technology we bring together genomes of different

organisms as the Genetic Code is universal the same proteins
that are encoded on Donor Genome will be formed in the
recepient.

P a g e 92
Monoclonal antibodies are specific to one antigene and
produced by identical immunce cells that are clones of unique
parent cell .

in 1975 Cesar Milsein, Georges Kohler, and Niels Kai Jerne

developed a technique for production of monoclonal antibodies.

P a g e 93
HAT medium (Hypoxanthine , aminopterin and thymidine) . this
medium is seclective only for Hybridoma cells (fusion of spleen
cells with myeloma cell) , However spleen and myeloma cells
die in this medium.

Myeloma cell without fusion can not grow and die because it
lacks HGPRT enzyme which is needed for Salvage Pathway
also they lost the ability of denovo synthesis because
Aminopterin inhibits Dihydrofolate reductase needed for denovo
synthesis of purines.

P a g e 94
Spleen cells have limited life span and die .

Hybridomas : Have HGPRT form spleen cells and can use

Salvage Pathway and the myeloma partner has traits that make it
immortal (similar to a cancer cell)so they produce large amounts
of monoclonal antibodies.

in 1984 they recieved Noble prize in medicine for their

technique.

In 1977 they discovered that genes in Eukaryotes contain

introns which are spliced in different ways to generate more
than one protein from same m-RNA.
The first mechanism: (self splicing)
1. It requires guanine nucleoside.
2. The 3-hydroxyl group of guanosine (found near 3`
end of intron) forms 3`-5` phosphodiester bond with
the 5` end of the intron,(1-3) . .(1st reaction)

P a g e 95
 3. The freed 3`- OH of exon 1 (1-4) attacks and forms a
phosphodiester bond with the 5 end of exon 2. (2nd
reaction)
4. The two exons are ligated and the introns are released .
The Second mechanism (splicosomal splicing):
1. This mechanism requires 5 small nuclear RNAs
(snRNAs) combine with protein, form small nuclear
ribonucleoprotein particles (snRNps). snRNps forms a
complex with a region on primary transcript called
splicosome. the binding of snRNps brings the
sequences of the neighboring exons into the correct
alignment for splicing.
2. The 2-OH of the Adenosine attacks and forms a
phosphodiester bond with 5 end of intron .(1st
reaction)
3. The freed 3`- OH of exon 1 attacks and forms a
phosphodiester bond with the 5 end of exon 2. (2nd
reaction)
4. The two exons are ligated and the introns are released .
In 1993 they shared Noble Prize for medicine.

P a g e 96
Do we care about these people that are HIV-positive whose lives
have been ruined? Those are the people I'm the most concerned
about. Every night I think about this.

Kary Mullis

He invented the PCR technique in 1983.

A complication at that point was that the DNA polymerase used

was destroyed by the high heat used at the start of each
replication cycle and had to be replaced.

In 1986, Saiki started to use thermophilus aquaticus bacterial

DNA polymerase (these bacteria live in hot springs 80 degree
celsius).

The Taq polymerase was heat resistant and would only need to
be added once, thus making the technique dramatically more
affordable and automatic.

P a g e 97
It is possible to increase a given segment of DNA in a short time
after an hour one obtaines millions to hundreds of millions
copies of the desired DNA .

This made it possible to obtain accurate analysis of DNA .

Forensic application of the technique have resulted in positive

identification of crime victims and suspects. Even ancient DNA
(Egyptian mummies ) can be analyzed..

In 1993 he won Noble Prize for invention of PCR.

P a g e 98
P a g e 99
 Further Reading

Lehninger, Albert (2008). Principles of Biochemistry.

New York, NY: W.H. Freeman and Company.
p. 539. ISBN 0-7167-7108-X.

Krebs, Hans (1972). "The Pasteur effect and the

relations between respiration and
fermentation". Essays in Biochemistry (8): 134.

Asimov, Asimov's Biographical Encyclopedia of Science and

Technology 2nd Revised edition

Athel Cornish-Bowden (1999). "The Origins of

Enzymology." // The Biochemist 19(2), 3638.
http://www.ncbi.nlm.nih.gov/books/NBK6243/
http://www.britannica.com/EBchecked/topic/392256/Thoma
s-Hunt-Morgan
http://www.dnaftb.org/10/bio.html
http://en.wikipedia.org/wiki/PH_meter#History
Chargaff, Erwin; Stephen Zamenhof; Charlotte Green (May
1950). "Composition of human desoxypentose nucleic acid".
Nature 165 (4202): 7567. Bibcode:1950Natur.165..756C.
doi:10.1038/165756b0. PMID 15416834.
Noble Prize website.
British Encyclopaedia.
Wikipaedia the free Encyclopaedia.
Cambpell biocehmistry.

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