Biosynthetic reactions of amino acids and Gel Electrophoresis

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◘ Biosynthetic reactions of amino acids
♦ Neurotransmitters
♣ Either Excitatory
• Excitatory neurotransmitters ( e.g. glytamate and acetylcholine ) promate
the depolarization of the membrane in another cell (either another neuron or
an effectors cell, such as a muscle cell) .
• If the postsynaptic cell is a muscle cell, sufficient release of excitatory
neurotransmitter molecules results in muscle contraction.
♣ Or inhibitory
• Inhibitory neurotransmitters ( e.g., glycine ) make the membrane potential
in the postsynaptic cell even more negative, that is, they inhibit the formation
of an action potential .
• A significant percentage of neurotransmitter molecules are either amino
acid or amino acid derivatives.
• The latter class is often referred to as the biogenic amines .
♣ Amino acids may acts as neurotransmitters
(1A) by decarboxylation using L-amino acid decarboxylase & B6P
- This is formed in the brain by decarboxylation of glutanic acid,where it
may act as a chemical mediator in the transmission of the nerve impulse
between some neurons .

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(1B) By decarboxylation using L-amino acid decarboxylase & B6P
- Histamin : arises from histidine by decarboxylation, It is a vasodilator and
is involved in shock and allergic responses .
♠ Histamin :
• It is a mediator of allergic and inflammatory reactions, a stimulator of
gastric acid production, and a neurotransmitter in several areas of the brain.
♣ Catecholamines act as neurotransmitters and hormones
♦ 2.The Catecholamines :
• The catrcholamines (dopamine, norepinephrine, and epinephrine) are
derivatives of tyrosine.
• Dopamine (D) and norepinephrine (NE) are utilized in the brain as
excitatory neurotransmitters .
• Outside the central nervous system, NE and epinephrine € are released
primarily from the adrenal medulla, as well as the peripheral nervous system

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♣ Tryptophan Derivatives :

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♦ 3. Serotonin :
• Serotonin is found in various cells within the central nervous system, where
it exerts an inhibitory effect on feeding behavior .
• Serotonin has been implicated in human eating disorders such as anorexia
nervosa, bulimia, and the carbohydrate craving associated with seasonal
affective disorder .
• Additionally, serotonin appears to affect mood, temperature regulation,
pain perception, and sleep.
• Serotonin is also found in the gastrointestinal tract, blood platelets, and
mast cells .
♦ 4. Melatonin
• Formed principally in pineal gland.
• Synthesis controlled by light, among other factors .
• Induces skin lightening .
• Suppresses ovarian function .
• Possible use in sleep disorders .
♣ Glycine
• Is important for heme synthesis.
• Prosthetic group of hemoglobin, myoglobin,
cytochromes, and hydroperoxidases .

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• Glycine Is Important for creatine synthesis

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• Creatine : Used for energy storage, e.g., in muscle, heart & brain
( Traces pass in the urine )
• Creatinine concentration is the plasma and creatinine in 24 hours urine are
constant and proportionate to muscle mass .
• Plasma creatinine concentration increases with impaired kidney function.
• Reference range = 0.6 – 1.4 mg/dl

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• Glycine is important for bile salts synthesis .
• Conjugation of bile acids with glycine or taurine ( derived from cysteine )
ensures higher ionization .
♠ Bile salts :
• Bile salts by their amphipathic nature are important for emulsification of
lipids :
- Digestion and absorption of fat and fat soluble vitamins .
◘ Working with proteins :
• A pure preparation is essential before a protein’s properties and activities
can be determined.
• Given that cells contain thousands of different kinds of proteins, how can
one protein are purified ?

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• Methods for separating proteins take advantage of properties that vary from
one protein to the next, including size, charge, and binding properties .
• For example, dialysis is a procedure that separates proteins from solvents
by taking advantage of the proteins’ larger size .
• Dialysis might be used, for example, to remove ammonium sulfate from the
protein preparation .
• The most powerful methods for fractionating proteins make use of
chromatographic techniques, which takes advantage of differences in protein
charge, size, binding affinity, and other properties .
◘ Separation Techniques :
• All separation procedures depend primarily upon some physical
characteristic of the compounds .
• It is relatively easy to separate substances, which have significantly
different physical characteristics by simple techniques, such as :
- Filtration
- Solvent extraction
- Evaporation
- Centrifugation
- Distillation
- Chromatography

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♦ What is chromatography ?
• It is a broad range of physical methods used to separate and or to analyze
complex mixtures .
• The components to be separated are distributed between two phases :
A Stationary phase and A Mobile phase which percolates through the
stationary phase .
• Molecules that spend most of their time in the mobile phase are carried
along faster .
• Chromatography is a Technique used for separating mixtures into their
components in order to Analyze, Identify, Purify, And/ Or Quantify the
Mixture of components .
♦ Uses For Chromatography :
• Analyze : examine a mixture, its components, and their relations to one
another .
• Identify : determine the mixture or components based on known
components .
• Purify : separate components in order to isolate one of interest for further
study .
• Quantify : determine the amount of the mixture and/or the components
present in the sample .

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♦ Real-life examples of uses for chromatography :
• Pharmaceutical company : determine amount of each chemical found in
new product .
• Hospital : detect blood or alcohol levels in a patient’s blood stream.
• Law Enforcement : to compare a sample found at a crime scene to
samples from suspects .
• Environmental Agency : determine the level of pollutants in the water
supply.
• Manufacturing plant : to purify a chemical needed to make a product .
♦ Gel permeation chromatography (GPC) :

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♣ Applications :
- Purifications .
- Solution Concentration .
- Desalting .
- Protein Binding Studies .
- Molecular Weight Determination .
◘ Electrophoresis :
• Electrophoresis is a method whereby charged molecules in solution, chiefly
proteins and nucleic acids, migrate in response to an electrical field .
• Their rate of migration through the electrical field, depends in the strength
of the field, on the net charge, size, and shape of the molecules, and also on
the ionic strength, viscosity, and temperature of the medium in which the
molecules are moving .
• Electrophoresis is usually done with gels formed in tubes, slabs, or on a flat
bed .
• In many electrophoresis units, the gel is mounted between two buffer
chambers containing separate electrodes, so that the only electrical
connection between the two chambers is through the gel .
• Separation of a mixture of charged molecules :
- Charged molecules are separated based on their electrical charge and size .

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- Charged molecules are separated based on their electrical charge and size .

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♦ Gel Electrophoresis
• Electrophoresis :
- Movement of charged particles through a solution (gel) under the influence
of an electric field .
• The Basic Components :
- The Gel in gel electrophoresis is the component that physically separates
the molecules .
- The Electric power supply provides the electricity that carries the
molecules through the gel .
- The Electrophoresis Chamber is where it all takes place .
- The gels in gel electrophoresis, however, retard large molecules relative to
smaller ones, the reverse of what occurs in gel filtration chromatography,
because there is no solvent space in gel electrophoresis analogous to that
between the gel beads in gel filtration chromatography .
• The process :
- Gels are often made of a gelatin-like substance called agarose .
- A comb with teeth is suspended in the gel until it cools .
- The gel is then placed in the electrophoresis chamber.
- Gel acts like a bridge through which the molecules must travel to get from
one side of the chamber to the next .
- After all the samples have been loaded into the wells, the electrodes are
connected to the power supply .
- When the power is turned on (Set power supply to about 80-100 Volts).

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- The negatively charged molecules move through the gel toward the positive
pole ( Remember: Opposite do attract !! ) .
- When the electrophoresis is complete, it is then time to stain the gel .

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♦ Silver stained gel
- Silver staining is usually 10-100 times more sensitive than Coomassie Blue
staining, but it is more complicated .
- Faint but still visible bands in this gel contain less than 0.5 ng of proteins! .