Download PHL 224 Biochemistry II

PHL 224 Biochemistry II
What are amino acids?
 Amino Acids are the building blocks of proteins. Proteins are polymers of amino acids linked together by what is called
“Peptide bond”.
 Twenty percent of the human body is made up of protein

There are about 300 amino acids occur in nature. Only 20 of them occur in proteins.
PROPERTIES OF AMINO ACIDS
Physical Properties of Amino acids:
Amino acids have a tetrahedral structure.
1. Structure & Optical property:
The α-carbon of an amino acid is attached to four different chemical groups and is, therefore, a chiral or optically active carbon
atom. Glycine is the exception because its α-carbon has two hydrogen substituents and, therefore, is optically inactive. Amino
acids that have an asymmetric center at the α-carbon can exist in two forms, designated D (Dextro-dextrorotatory) and L (Levo-
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PHL 224 Biochemistry II
levorotatory) that are mirror images of each other. The two forms in each pair are termed stereoisomers, optical
isomers, or enantiomers. All amino acids found in proteins are of the L-configuration. However, D-amino acids are found in some
antibiotics and in plant and bacterial cell walls
Figure: Mirror imaging of optical isomers of amino acids
Zwitterion: At pH 7 amino group is protonated (-NH3+ )
and carboxylic group is ionized (COO-)
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PHL 224 Biochemistry II
2. Solubility: Most of the amino acids are usually soluble in water and insoluble in organic solvents.
3. Melting points: Amino acids generally melt at higher temperatures, often above 200°C.
4. Taste: Amino acids may be sweet (Gly, Ala, Val), tasteless (Leu) or bitter (Arg, lle). Monosodium glutamate is used as a flavoring
agent in food industry, and Chinese foods to increase taste and flavor.
Importance of amino acids
Amino acids have an influence on the function of organs, glands, tendons and arteries. They are furthermore essential for healing
wounds and repairing tissue, especially in the muscles, bones, skin and hair as well as for the removal of all kinds of waste deposits
produced in connection with the metabolism.
Essential Amino Acids: (cannot be synthesized by an organism and must be obtained in the diet) Histidine, Isoleucine, Leucine,
Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine.
Non-essential Amino Acids: Alanine, Asparagine, Aspartic Acid, Glutamic Acid.
Conditional Amino Acids: Arginine (essential in children, not in adults), Cysteine, Glutamine, Glycine, Proline, Serine, and Tyrosine.
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PHL 224 Biochemistry II
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PHL 224 Biochemistry II
In protein formation, the condensation of amino group of one α-amino acid with the carboxyl group of another molecule of
same or different α-amino acid with the elimination of a water molecule forms an amide linkage between molecules. This
amide linkage (-C(=O)NH-) between two α-amino acids is termed as peptide bond or peptide linkage. The dimer formed due to
this linkage is called as dipeptide.
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PHL 224 Biochemistry II
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PHL 224 Biochemistry II
Amino acid
3-letter
1-letter
abbreviation
code
Gly
G
Glycine
Properties
Structure
Optically inactive -Conditional amino acid
colourless, sweet-tasting crystalline solid
Source:biosynthesized in the body from the amino acid serine.
Function:precursor to proteins, such as Collagen helix in conjunction with
Hydroxyproline,Used in: Certain drug formulations to improve gastric
absorption of the drug, and additive in pet food . For humans, as a
sweetener/taste enhancer. In certain food supplements and protein drinks
Ala
Alanine
Non-polar - Non-essential amino acid
A
Found in: animal sources: meat, seafood, eggs, fish and gelatin. Vegetarian
sources: beans, nuts, seeds, soy, whey, brewer's yeast, brown rice, bran, corn
and whole grains.
Function: plays a key role in glucose–alanine cycle between tissues and liver
Phe
Phenylalanine
Non-polar- Essential amino acid
F
Found naturally in the breast milk of mammals used as dietary supplement
Function: Precursor to Tyrosine
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PHL 224 Biochemistry II
Glutamic acid
Acidic - Non-essential amino acid
Glu
E
Found In All meats, poultry, fish, eggs, dairy products and wheat
Function: Glutamate is a key compound in cellular metabolism
Used as a food additive and flavor enhancer
Tyrosine
Conditional Amino Acid
Tyr
Y
Found in chicken, turkey, fish, milk, yogurt, cottage cheese, cheese, peanuts,
almonds, pumpkin seeds, sesame seeds, soy products, lima beans, avocados,
and bananas.
Function: Precursor to neurotransmitters (L-Dopa), alkaloids(morphine)and
pigment melanin
Tryptophan
Trp
Essential Amino Acid
W
Found in chocolate, oats, dried dates, milk, yogurt, cottage cheese, red meat,
eggs, fish, poultry, sesame, chickpeas, sunflower seeds, pumpkin seeds,
spirulina, bananas, and peanuts
Function: Precursor to neurotransmitters Serotonin
Methionine
Met
Essential Amino Acid
M
Found in eggs, sesame seeds, Brazil nuts, fish, meats and some plant seeds.
Function: intermediate in the biosynthesis of cysteine, carnitine, taurine,
lecithin, phosphatidylcholine, and other phospholipids.
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PHL 224 Biochemistry II
(Hydrolyzed collagen )
Gelatin
(Hydrolyzed collagen )
Bone, skin&
connective
tissue.
Albumin
Solubility
Uses
Hot water, and sets to a gel on cooling. If
Improving hair quality, the shells of
added directly to cold water does not
pharmaceutical capsules in order to make
dissolve well. Also soluble in most polar
them easier to swallow ,also preparation
solvents
of foods, cosmetics.
found in
Protein
main protein in human blood
Function is to regulate the colloidal
produced in the liver
blood& egg
Reference range 35 - 50 g/L
white
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Water
osmotic pressure of blood. and Transports
hormones, fatty acids
PHL 224 Biochemistry II
1- Solubility Tests:
Principle:
The solubility of amino acids and proteins is largely dependent on the solution pH. The structural changes in an amino acid or
protein that take place at different pH values alter the relative solubility of the molecule. In acidic solutions, both amino and
carboxylic groups are protonated. In basic solutions, both groups are deprotonated.
Amino acids are essentially soluble in water. Their solubilities in water, dilute alkali and dilute acid vary from one compound to the
other depending on the structure of their side chains. Apply this test to glycine, tyrosine, glutamic acid and cysteine.
Procedure:
1- Note the solubility of amino acids in water and alcohol by placing a small amount in a test tube, adding a few mL of solvent and
warming if necessary.
2- Determine the amino acid solution is acidic or basic by using a litmus paper while testing the solubility in water.
3- Repeat the solubility test using dilute HCl and dilute NaOH.
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PHL 224 Biochemistry II
Experimental Procedure:
No.
Test
I.
1.
Observation
Inference & Interpretation
General Test for amino acid and protein (Amine groups in proteins, peptones and amino acids)
Ninhydrin test:
a- Protein or Amino acid present
Principle: In the pH range of 4-8, all α- amino acids react
a- blue color formed
(primary amine)
with ninhydrin (triketohydrindene hydrate), a powerful
detect alpha-amino acids and also free amino
and carboxylic acid groups on proteins and
oxidizing agent to give colored product (diketohydrin)
peptides
termed Rhuemann’s purple.
Ninhydrin is most commonly used as a
forensic chemical to detect “fingerprints”, as
Procedure: To 1ml solution add 5 drops of 0.2% ninhydrin
amines left over from proteins sloughed off
solution in acetone. Boil over a water bath for 2 min. Allow
in fingerprints react with ninhydrin giving a
to cool.
characteristic purple color.
N.B: Avoid spilling ninhydrin solutions on your skin, as the
resulting stains are difficult to remove.
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b. yellow color is formed
(secondary amine)
Prepared by Lecturer. Ghada Fekry.
b- Presence of amino acid Proline
PHL 224 Biochemistry II
II. Tests for protein
1.
Biuret’s test
Principle: Biuret test is Specific for Proteins differentiate
between Proteins (+ve) and Amino Acids (-ve). The biuret
reagent (copper sulfate in a strong base) reacts with
Violet color is obtained with
albumin, casein & gelatin
and a pinkish violet color with
peptone
Protein
Two peptide bonds are at least required for
peptide bonds in proteins to form a violet complex known
the formation of this complex , this is why
as “Biuret complex”.
amino acids give negative results with Biuret
Procedure :To 1 mL of protein solution (Albumin – Casein –
test.
Gelatin – Peptone) in a test tube, add 1 mL of 10% sodium
hydroxide solution and 2-3 drops of 1% copper sulfate
solution. Mix well
2.
Heat coagulation test
Principle: protein coagulated by heating, acetic acid is
added, if coagulation persists, its protein.
Procedure Place about 5 ml of egg-white solution (albumin
cloudy and a flocculent precipitate of
coagulated protein is produced
solution) in a test tube and heat the top part of the solution
only.
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Protein
PHL 224 Biochemistry II
3.
Picric acid test
Procedure To 3 ml of gelatin solution in a test tube, add
Protein
2ml of saturated picric acid solution
a
yellow gelatinous precipitate
4.
Precipitation by salts of heavy metal:
Principle: protein precipitate by heavy metals, where
positively charged metal ion neutralize the negatively
charged protein molecule resulting in
Metal proteinate.
Protein
White precipitate
A- Procedure: to protein solution add mercuric chloride
drop by drop.
B- Procedure: to protein solution add lead acetate soln
drop by drop.
III. Test for α -amino-acid glycine
1.
p-nitrobenzoyl chloride & pyridine test:
orange-red
to
maroon
color
Glycine
Principle: Glycine in quantities as small as 0.5 g detected as develops immediately, varying in N.B. As little as 0.5 g. of glycine develops a
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PHL 224 Biochemistry II
an orange-red color by reaction with p-nitrobenzoyl shade with the concentration
perceptible orange-red color. All other amino
chloride & pyridine. The procedure can be used both as a of glycine.
acids fail to react in concentrations below 0.5
qualitative spot test and as a sensitive quantitative method,
mg with larger quantities, a pale bluish color,
the color is due to aziactone formation.
in contrast to colorless blanks, develops with
Procedure: A few crystals of powder sample, on a filter
some amino acids.
paper strip or a glass slide, and approximately 1 mg. of solid
Acetylglycine, glycyiglycine, hippuric acid,
p-nitrobenzoyl chloride is placed on top. One to three drops
and salicyluric acid do not react in the cold,
of pyridine are then added to wet the mixture.
but on warming, a yellow color is produced
The color is soluble in polar solvents such as chloroform,
with milligram quantities.
dichioroethylene, tetrachloroethane, ethylacetate, and also
in excess pyridine.
IV. Test for amino-acids contain: activated benzene rings ( Tyrosine and Tryptophan)
1.
Xanthoproteic test:
Yellow
to
Orange
Principle: Aromatic amino acids, such as tyrosine and
tryptophan, respond to this test. In the presence of
concentrated nitric acid, the aromatic phenyl ring is nitrated
to give yellow colored nitro-derivatives. At alkaline pH, the
color changes to orange due to the ionization of the
phenolic group.
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Amino acid present
PHL 224 Biochemistry II
procedure: To 2ml of solution in a test tube, add an equal
(tyrosine, tryptophan)
volume of conc. HNO3. Heat over a flame for 2 min and
observe the color. Now COOL THOROUGHLY under the tap
and CAUTIOSLY run in sufficient 40% NaOH to make the
solution strongly alkaline.
V. Test for amino-acids contain: Indol group
1.
Hopkins-Cole Test:
Principle:This test is specific test for detecting tryptophan.
purple color at the interface
The indole moiety of tryptophan reacts with glyoxilic acid in
the presence of concentrated sulphuric acid to give a purple
Amino acid present
colored product. Glyoxilic acid is prepared from glacial
Tryptophan
acetic acid by being exposed to sunlight.
Procedure: To a few ml of glacial acetic acid containing
glyoxylic acid, add 1-2 drops of the amino acid solution.
Pour 1-2ml concentrated H2SO4 down the side of the
sloping test tube to form a layer underneath the acetic acid.
2.
Ehrlich's test: To 0.5ml of the amino acid solution, add
A colored complex formed
Tryptophan
2ml Ehrlich reagent
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Amino acid present
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PHL 224 Biochemistry II
VI. Test for : phenolic amino acid
1.
Millon’s test:
A brick
red color is a positive
Principle: Phenolic amino acids such as Tyrosine and its
reaction.
derivatives respond to this test. Compounds with a
(red – pink colour )
hydroxybenzene radical react with Millon’s reagent to form
Amino acid present
phenolic amino acid as (tyrosine)
a red colored complex. Millon’s reagent is a solution of
mercuric sulphate in sulphuric acid.
N.B: A yellow precipitate of HgO is NOT a
Procedure: To 2ml of amino acid solution in a test tube, add
positive reaction but usually indicates that
1-2 drops of Millon’s reagent. Warm the tube in a boiling
the solution is too alkaline.
bath
VII. Test for amino-acids contain: Sulfhydryl group –SH (cystine &Cysteine)
1.
Nitroprusside test:
Amino acid present
Principle It is specific for Amino Acids or Proteins containing
sulfur, -SH (in cysteine & cystine)
Red color formed
Procedure:Add 2ml of the amino acid solution into test
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(cystine, cysteine )
The nitroprusside test is specific for cystine &
PHL 224 Biochemistry II
tubes. Add 0.5ml fresh sodium nitroprusside solution and
cysteine, the only amino acid containing
shake thoroughly. Add 0.5ml ammonium hydroxide.
sulfhydryl group (-SH).
VIII. Test for amino-acids contain: Sulfur
1.
Lead acetate test:
Principle:cysteine and cystine, the sulfur containing amino
acid react with lead acetate under alkaline conditions to
form a brown precipitate .
Procedure: Everything needed to carry out this test will be
in the hood and should not remove anything from the hood.
Amino acid containing sulphur present
A toxic, stinky gas will be made (in small, but immensely
(cystine , cysteine )
smelly quantities) and you don’t want to smell it. Dispense
about 0.5mL of the amino acid solution only into a clean
test tube (found in the hood, where you will leave it when
brownish-black precipitate
you are done). Add 0.5mL of 20% NaOH and insert the test
tube in a boiling water bath for 1 min. Add 2 drops of lead
(II) acetate solution.
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PHL 224 Biochemistry II
IX. Test for amino-acids contain: Guanidium group
1.
Sakaguchi's test:
Red color formed
Principle Arginine, containing guanidine group reacts with
α -naphthol
under alkaline conditions to produce red
Amino acid containing guanidine present
color.
(Arginine)
Procedure: 1ml NaOH and 3ml of the arginine solution is
mixed and 2 drops of α-naphthol is added. Mix thoroughly
and add 4-5 drops Bromine solution
X. Test for Histadine amino-acids
1.
Pauly’s test:
Principle: This test is specific for the detection of Histidine.
The reagent used for this test contains sulphanilic acid
dissolved in hydrochloric acid. Sulphanilic acid upon
diazotization in the presence of sodium nitrite and
Yellow product formed
hydrochloric acid results in the formation a diazonium salt.
The diazonium salt formed couples with either tyrosine or
histidine in alkaline medium to give a red colored
chromogen (azo dye).
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Amino acid present
Histidine
PHL 224 Biochemistry II
Procedure: Into clean test tube, dispense 1mL of 1%
sulphanilic acid and 2 drops of 5% sodium nitrite. Mix for 1
min. Add about 0.5mL of amino acid solution.
Results: The protein or amino acids were found to be present in given sample.
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PHL 224 Biochemistry II
Ninhydrin
p-nitrobenzoyl chloride
Xanthoproteic
Hopkins-Cole
Ehrlich's
Test
& pyridine Test
Test
Test
Test
Glycine
+ve
+ve
Alanine
+ve
Phenylalanine
+ve
Glutamic acid
+ve
Tyrosine
+ve
+ve
Tryptophan
+ve
+ve
Methionine
+ve
AMINO acid
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Millon's Test
+ve
+ve
+ve