Upload
nguyenlien
View
229
Download
3
Embed Size (px)
Citation preview
PHL 224 Biochemistry II
30 Prepared by Lecturer. Ghada Fekry.
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-
PHL 224 Biochemistry II
31 Prepared by Lecturer. Ghada Fekry.
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-)
PHL 224 Biochemistry II
32 Prepared by Lecturer. Ghada Fekry.
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.
PHL 224 Biochemistry II
33 Prepared by Lecturer. Ghada Fekry.
PHL 224 Biochemistry II
34 Prepared by Lecturer. Ghada Fekry.
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.
PHL 224 Biochemistry II
35 Prepared by Lecturer. Ghada Fekry.
PHL 224 Biochemistry II
36 Prepared by Lecturer. Ghada Fekry.
Amino acid 3-letter
abbreviation
1-letter
code Structure
Properties
Glycine Gly G
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
Alanine Ala A
Non-polar - Non-essential amino acid
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
Phenylalanine Phe F
Non-polar- Essential amino acid
Found naturally in the breast milk of mammals used as dietary supplement
Function: Precursor to Tyrosine
PHL 224 Biochemistry II
37 Prepared by Lecturer. Ghada Fekry.
Glutamic acid Glu
E
Acidic - Non-essential amino acid
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 Tyr
Y
Conditional Amino Acid
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 W
Essential Amino Acid
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 M
Essential Amino Acid
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.
PHL 224 Biochemistry II
38 Prepared by Lecturer. Ghada Fekry.
Protein found in Solubility Uses
Gelatin (Hydrolyzed collagen )
(Hydrolyzed collagen )
Bone, skin&
connective
tissue.
Hot water, and sets to a gel on cooling. If
added directly to cold water does not
dissolve well. Also soluble in most polar
solvents
Improving hair quality, the shells of
pharmaceutical capsules in order to make
them easier to swallow ,also preparation
of foods, cosmetics.
Albumin
main protein in human blood
produced in the liver
Reference range 35 - 50 g/L
blood& egg
white Water
Function is to regulate the colloidal
osmotic pressure of blood. and Transports
hormones, fatty acids
PHL 224 Biochemistry II
39 Prepared by Lecturer. Ghada Fekry.
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.
PHL 224 Biochemistry II
40 Prepared by Lecturer. Ghada Fekry.
Experimental Procedure:
No. Test Observation Inference & Interpretation
I. General Test for amino acid and protein (Amine groups in proteins, peptones and amino acids)
1. Ninhydrin test:
Principle: In the pH range of 4-8, all α- amino acids react
with ninhydrin (triketohydrindene hydrate), a powerful
oxidizing agent to give colored product (diketohydrin)
termed Rhuemann’s purple.
Procedure: To 1ml solution add 5 drops of 0.2% ninhydrin
solution in acetone. Boil over a water bath for 2 min. Allow
to cool.
N.B: Avoid spilling ninhydrin solutions on your skin, as the
resulting stains are difficult to remove.
a- blue color formed (primary amine)
b. yellow color is formed (secondary amine)
a- Protein or Amino acid present
detect alpha-amino acids and also free amino
and carboxylic acid groups on proteins and
peptides
Ninhydrin is most commonly used as a
forensic chemical to detect “fingerprints”, as
amines left over from proteins sloughed off
in fingerprints react with ninhydrin giving a
characteristic purple color.
b- Presence of amino acid Proline
PHL 224 Biochemistry II
41 Prepared by Lecturer. Ghada Fekry.
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
peptide bonds in proteins to form a violet complex known
as “Biuret complex”.
Procedure :To 1 mL of protein solution (Albumin – Casein –
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
Violet color is obtained with albumin, casein & gelatin
and a pinkish violet color with peptone
Protein
Two peptide bonds are at least required for
the formation of this complex , this is why
amino acids give negative results with Biuret
test.
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
solution) in a test tube and heat the top part of the solution
only.
cloudy and a flocculent precipitate of coagulated protein is produced
Protein
PHL 224 Biochemistry II
42 Prepared by Lecturer. Ghada Fekry.
3. Picric acid test
Procedure To 3 ml of gelatin solution in a test tube, add
2ml of saturated picric acid solution
a
yellow gelatinous precipitate
Protein
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.
A- Procedure: to protein solution add mercuric chloride
drop by drop.
B- Procedure: to protein solution add lead acetate soln
drop by drop.
White precipitate Protein
III. Test for α -amino-acid glycine
1. p-nitrobenzoyl chloride & pyridine test:
Principle: Glycine in quantities as small as 0.5 g detected as
orange-red to maroon color
develops immediately, varying in
Glycine
N.B. As little as 0.5 g. of glycine develops a
PHL 224 Biochemistry II
43 Prepared by Lecturer. Ghada Fekry.
an orange-red color by reaction with p-nitrobenzoyl
chloride & pyridine. The procedure can be used both as a
qualitative spot test and as a sensitive quantitative method,
the color is due to aziactone formation.
Procedure: A few crystals of powder sample, on a filter
paper strip or a glass slide, and approximately 1 mg. of solid
p-nitrobenzoyl chloride is placed on top. One to three drops
of pyridine are then added to wet the mixture.
The color is soluble in polar solvents such as chloroform,
dichioroethylene, tetrachloroethane, ethylacetate, and also
in excess pyridine.
shade with the concentration
of glycine.
perceptible orange-red color. All other amino
acids fail to react in concentrations below 0.5
mg with larger quantities, a pale bluish color,
in contrast to colorless blanks, develops with
some amino acids.
Acetylglycine, glycyiglycine, hippuric acid,
and salicyluric acid do not react in the cold,
but on warming, a yellow color is produced
with milligram quantities.
IV. Test for amino-acids contain: activated benzene rings ( Tyrosine and Tryptophan)
1. Xanthoproteic test:
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.
Yellow to Orange
Amino acid present
PHL 224 Biochemistry II
44 Prepared by Lecturer. Ghada Fekry.
procedure: To 2ml of solution in a test tube, add an equal
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.
(tyrosine, tryptophan)
V. Test for amino-acids contain: Indol group
1. Hopkins-Cole Test:
Principle:This test is specific test for detecting tryptophan.
The indole moiety of tryptophan reacts with glyoxilic acid in
the presence of concentrated sulphuric acid to give a purple
colored product. Glyoxilic acid is prepared from glacial
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.
purple color at the interface
Amino acid present
Tryptophan
2. Ehrlich's test: To 0.5ml of the amino acid solution, add
2ml Ehrlich reagent
A colored complex formed
Amino acid present
Tryptophan
PHL 224 Biochemistry II
45 Prepared by Lecturer. Ghada Fekry.
VI. Test for : phenolic amino acid
1. Millon’s test:
Principle: Phenolic amino acids such as Tyrosine and its
derivatives respond to this test. Compounds with a
hydroxybenzene radical react with Millon’s reagent to form
a red colored complex. Millon’s reagent is a solution of
mercuric sulphate in sulphuric acid.
Procedure: To 2ml of amino acid solution in a test tube, add
1-2 drops of Millon’s reagent. Warm the tube in a boiling
bath
A brick red color is a positive
reaction.
(red – pink colour )
Amino acid present
phenolic amino acid as (tyrosine)
N.B: A yellow precipitate of HgO is NOT a
positive reaction but usually indicates that
the solution is too alkaline.
VII. Test for amino-acids contain: Sulfhydryl group –SH (cystine &Cysteine)
1. Nitroprusside test:
Principle It is specific for Amino Acids or Proteins containing
sulfur, -SH (in cysteine & cystine)
Procedure:Add 2ml of the amino acid solution into test
Red color formed
Amino acid present
(cystine, cysteine )
The nitroprusside test is specific for cystine &
PHL 224 Biochemistry II
46 Prepared by Lecturer. Ghada Fekry.
tubes. Add 0.5ml fresh sodium nitroprusside solution and
shake thoroughly. Add 0.5ml ammonium hydroxide.
cysteine, the only amino acid containing
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.
A toxic, stinky gas will be made (in small, but immensely
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
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.
brownish-black precipitate
Amino acid containing sulphur present
(cystine , cysteine )
PHL 224 Biochemistry II
47 Prepared by Lecturer. Ghada Fekry.
IX. Test for amino-acids contain: Guanidium group
1. Sakaguchi's test:
Principle Arginine, containing guanidine group reacts with
α -naphthol under alkaline conditions to produce red
color.
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
Red color formed
Amino acid containing guanidine present
(Arginine)
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
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).
Yellow product formed Amino acid present
Histidine
PHL 224 Biochemistry II
48 Prepared by Lecturer. Ghada Fekry.
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.
PHL 224 Biochemistry II
49 Prepared by Lecturer. Ghada Fekry.
AMINO acid Ninhydrin
Test
p-nitrobenzoyl chloride
& pyridine Test
Xanthoproteic
Test
Hopkins-Cole
Test
Ehrlich's
Test Millon's Test
Glycine +ve +ve
Alanine +ve
Phenylalanine +ve
Glutamic acid +ve
Tyrosine +ve +ve +ve
Tryptophan +ve +ve +ve +ve
Methionine +ve