JAHANGIRNAGAR UNIVERSITY

Department of Biochemistry & Molecular Biology

Assignment title: Amino Acid

Course Title: Introductory Biochemistry
Course No: BMB 101
Date of Submission: June 17, 2021

Submitted To:
Farha Matin Juliana
Professor,
Department of Biochemistry & Molecular Biology,
Jahangirnagar University

Submitted By:

Shanian Ahmed
Roll: 1528
Batch: 49
Department of Biochemistry & Molecular Biology
Jahangirnagar University

1|Page
Contents
Introduction .................................................................................................................................................. 3
Structure of amino acids ............................................................................................................................... 3
Classification of amino acids ......................................................................................................................... 4
Functional group of amino acids................................................................................................................. 11
Standard amino acids.................................................................................................................................. 11
Nonstandard amino acids ........................................................................................................................... 12
L and D amino acids .................................................................................................................................... 14
Properties of Amino Acids .......................................................................................................................... 15
Some common uses of amino acids............................................................................................................ 18
Biomedical Importance of amino acids....................................................................................................... 19
Commercial Importance of amino acids ..................................................................................................... 20
Identification of amino acids ...................................................................................................................... 22

2|Page
Introduction
An amino acid is a type of organic acid that contains a carboxyl functional group (-
COOH) and an amine functional group (-NH2) as well as a side chain (designated as
R) that is specific to the individual amino acid. The elements found in all amino acids
are carbon, hydrogen, oxygen, and nitrogen, but their side chains may contain
other elements as well.

Shorthand notation for amino acids may be either a three-letter abbreviation or a

single letter. For example, valine may be indicated by V or val; histidine is H or his.

Amino acids may function on their own, but more commonly act as monomers to
form larger molecules. Linking a few amino acids together forms peptides, and a
chain of many amino acids is called a polypeptide. Polypeptides may be modified
and combine to become proteins.

Structure of amino acids

Generally, amino acids have the following structural properties:

A carbon (the alpha carbon)

A hydrogen atom (H)

A Carboxyl group (-COOH)

An Amino group (-NH2)

A "variable" group or "R" group

3|Page
All amino acids have the alpha carbon bonded to a hydrogen atom, carboxyl group,
and amino group. The "R" group varies among amino acids and determines the
differences between these protein monomers. The amino acid sequence of a
protein is determined by the information found in the cellular genetic code. The
genetic code is the sequence of nucleotide bases in nucleic acids (DNA and RNA)
that code for amino acids. These gene codes not only determine the order of amino
acids in a protein, but they also determine a protein's structure and function.

Classification of amino acids

I. Chemical Classification: According to number of COOH and NH2 groups i.e.
according to net charge on amino acid.

A- Monobasic, monocarboxylic amino acids i.e. neutral or uncharged:

Sub-classification of neutral amino acids:

1- Glycine R= H

2- Alanine R=CH3

3- Branched chain amino acids: R is branched such as in:

a- Valine R= isopropyl gp

b- Leucine R= isobutyl gp

c- Isoleucine R= is isobutyl

R is isobutyl in both leucine and isoleucine but branching is different.

in Leucine – branching occurs on Gama carbon

in isoleucine – branching occurs in Beta carbon

4- Neutral Sulfur containing amino acids:

e.g. Cysteine and Methionine

4|Page
5- Neutral, hydroxyl amino acids:

e.g. Serine and Threonine

6- Neutral aromatic amino acids:

e.g. Phenyl alanine and Tyrosine

7- Neutral heterocyclic amino acids:

e.g. Tryptophan and Proline

B- Basic amino acids:

Basic amino acids contain two or more NH2 groups or nitrogen atoms. The three
amino acids in this group are arginine, histidine, and lysine. Each side chain is basic
(i.e. can accept a proton). Lysine and arginine both exist with an overall charge of
+1 at physiological pH. The guanido group in arginine’s side chain is the most basic
of all R groups. As mentioned above for aspartate and glutamate, the side chains
of arginine and lysine also form ionic bonds. The chemical structures of basic amino
acids are:

5|Page
C- Acidic amino acids:

The two amino acids in this group are aspartic acid and glutamic acid. Each has a
carboxylic acid on its side chain that gives it acidic (proton-donating) properties. In
an aqueous solution at physiological pH, all three functional groups on these amino
acids will ionize, thus giving an overall charge of −1. In the ionic forms, the amino
acids are called aspartate and glutamate. The chemical structures of acidic amino
acids are:

The side chains of aspartate and glutamate can form ionic bonds (“salt bridges”),
and they can also function as hydrogen bond acceptors. Many proteins that bind
metal ions for structural or functional purposes possess metal-binding sites
containing aspartate or glutamate side chains or both. Free glutamate and
glutamine play a central role in amino acid metabolism. Glutamate is the most
abundant excitatory neurotransmitter in the central nervous system.

6|Page
II. Classification according to polarity of side chain (R):
A- Polar amino acids:

Polar amino acids are serine, cysteine, threonine, tyrosine, asparagine, and
glutamine. The side chains in this group possess a spectrum of functional groups.

However, most have at least one atom (nitrogen, oxygen, or sulfur) with electron
pairs available for hydrogen bonding to water and other molecules. The chemical
structures of polar amino acids are:

Two amino acids, serine and threonine, contain aliphatic hydroxyl groups (that is,
an oxygen atom bonded to a hydrogen atom, represented as ―OH). Tyrosine
possesses a hydroxyl group in the aromatic ring, making it a phenol derivative. The
hydroxyl groups in these three amino acids are subject to an important type of

7|Page
posttranslational modification: phosphorylation (see below Nonstandard amino
acids). Like methionine, cysteine contains a sulfur atom. Unlike methionine’s sulfur
atom, however, cysteine’s sulfur is very chemically reactive (see below Cysteine
oxidation). Asparagine, first isolated from asparagus, and glutamine both contain
amide R groups. The carbonyl group can function as a hydrogen bond acceptor, and
the amino group (NH2) can function as a hydrogen bond donor.

B- Nonpolar amino acids:

Nonpolar amino acids are glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, and tryptophan. The R groups of these amino acids
have either aliphatic or aromatic groups. This makes them hydrophobic (“water
fearing”). In aqueous solutions, globular proteins will fold into a three-dimensional
shape to bury these hydrophobic side chains in the protein interior. Isoleucine is an
isomer of leucine, and it contains two chiral carbon atoms. Proline is unique among
the standard amino acids in that it does not have both free α-amino and free α-
carboxyl groups. Instead, its side chain forms a cyclic structure as the nitrogen atom
of proline is linked to two carbon atoms. (Strictly speaking, this means that proline
is not an amino acid but rather an α-imino acid.) Phenylalanine, as the name
implies, consists of a phenyl group attached to alanine. Methionine is one of the
two amino acids that possess a sulfur atom. Methionine plays a central role in
protein biosynthesis (translation) as it is almost always the initiating amino acid.
Methionine also provides methyl groups for metabolism. Tryptophan contains an
indole ring attached to the alanyl side chain. The chemical structures of nonpolar
amino acids are:

8|Page
9|Page
III. Nutritional classification:
1. Essential amino acids:

These amino acids are not synthesized in cells of human beings, so these should be
essentially present in diet. Phenylalanine, Valine, Threonine, Tryptophan,
Isoleucine, Methionine, Leucine and Lysine are essential amino acids. Their
deficiency affects growth, health and protein synthesis.

2. Semi-essential amino acids:

These are formed in the body but not in sufficient amount for body requirements
especially in children. Arginine and Histidine are semi-essential.

3. Nonessential amino acids:

These amino acids can be synthesized in body, so need not be included in diet.
Glycine, Alanine, Serine, Cysteine, Asparagine, Glutamine, Aspartic acid, Glutamic
acid, Tyrosine and Proline are nonessential amino acids.

IV. Metabolic classification:

1. Glucogenic amino acids:

These amino acids serve as precursors gluconeogenesis for glucose formation

Glycine, Alanine, methionine and Aspartic acid are glucogenic amino acids.

2. Ketogenic amino acids:

These amino acids breakdown to form ketone bodies. Leucine and Lysine are
ketogenic amino acids.

3. Both glucogenic and ketogenic amino acids:

These amino acids breakdown to form precursors for both ketone bodies and
glucose. These are Isoleucine, Phenylalanine, Tryptophan and Tyrosine.

10 | P a g e
Functional group of amino acids
As briefly discussed earlier, amino acid functional groups determine the type or
class of amino acid present. The molecular structure of these functional groups are
usually represented by the placeholder labeled “R”. This specific placeholder serves
as an indicator where the functional group will be located. These functional groups
are in turn, connected to a carbon atom and to the right and left of this carbon
atom, also identified as the backbone of the amino acid, are the carboxylic acid
(COOH) and amine group (NH2). This design and structure comprises what is called
an amino acid. These amino acids will have their functional groups and their unique
properties composed of molecular structures that may be as simple as hydrogen or
as large as a circular molecular structure. There are three (3) Major types of
Functional Groups based on the R group of the said amino acid. These are:

1. Amino Acids with Nonpolar side chains,

2. Amino Acids with Polar Side Chains and

3. Amino Acids that have electrically charged side chains.

Standard amino acids

A standard amino acid is an amino acid organisms use in the synthesis of peptides.
There are twenty standard amino acids, all of which are alpha amino acids. Each
standard amino acid has a three-letter symbol and a one-letter symbol. Nineteen
of the twenty standard amino acids are chiral, and only the L isomer (see D, L
convention) occurs in eukaryotic. The standard amino acids are:

- glycine

- alanine

- proline

- valine

11 | P a g e
- leucine

- isoleucine

- methionine

- phenylalanine

- tyrosine

- tryptophan

- serine

- threonine

- cysteine

- asparagine

- glutamine

- lysine

- arginine

- histidine

- aspartate

- glutamate

Nonstandard amino acids

Nonstandard amino acids refer to those amino acids that have been chemically
modified after they have been incorporated into a protein (called a
“posttranslational modification”) and those amino acids that occur in living
organisms but are not found in proteins. Among these modified amino acids is γ-
carboxyglutamic acid, a calcium-binding amino acid residue found in the blood-
clotting protein prothrombin (as well as in other proteins that bind calcium as part

12 | P a g e
of their biological function). The most abundant protein by mass in vertebrates is
collagen. Significant proportions of the amino acids in collagen are modified forms
of proline and lysine: 4-hydroxyproline and 5-hydroxylysine.

Arguably, the most important posttranslational modification of amino acids in

eukaryotic organisms (including humans) is the reversible addition of a phosphate
molecule to the hydroxyl portion of the R groups of serine, threonine, and tyrosine.
This event is known as phosphorylation and is used to regulate the activity of
proteins in their minute-to-minute functioning in the cell. Serine is the most
commonly phosphorylated residue in proteins, threonine is second, and tyrosine is
third.

Proteins with carbohydrates (sugars) covalently attached to them are called

glycoproteins. Glycoproteins are widely distributed in nature and provide the
spectrum of functions already discussed for unmodified proteins. The sugar groups
in glycoproteins are attached to amino acids through either oxygen (O-linked
sugars) or nitrogen atoms (N-linked sugars) in the amino acid residues. The O-linked
sugars are attached to proteins through the oxygen atoms in serine, threonine,
hydroxylysine, or hydroxylproline residues. The N-linked sugars are attached to
proteins through the nitrogen atom in asparagine.

Finally, there is the case of selenocysteine. Although it is part of only a few known
proteins, there is a sound scientific reason to consider this the 21st amino acid
because it is in fact introduced during protein biosynthesis rather than created by
a posttranslational modification. Selenocysteine is actually derived from the amino
acid serine (in a very complicated fashion), and it contains selenium instead of the
sulfur of cysteine.

13 | P a g e
L and D amino acids
L- and D-amino acids are the two isomeric forms of amino acids which occur in
nature. L-amino acids are the form used by the cells to synthesize proteins.

What is L amino acid:

L-amino acids are the form of stereoisomers used by the cells to produce proteins.
They occur in all proteins produced by animals, plants, fungi, and bacteria. These
proteins have both structural and functional roles inside the cell. They serve as
enzymes, which catalyze biochemical reactions, as well as hormones, which
regulate the biological process in higher organisms. Also, they are the form of
amino acids produced by lightning reactions as well. In the Fisher projection, the
amine group of these amino acids occurs on the left side.

What is D amino acid:

D-amino acids are the other form of stereoisomers that occur in nature. The amine
group of these amino acids occurs in the right side in the Fisher projection.
Generally, cells do not incorporate D-amino acids into proteins. But, some proteins
are produced by enzyme posttranslational modifications in cone snails. On the
other hand, some D-amino acids also occur in the peptidoglycan cell walls of
bacteria. In addition, D-serine serves as a neurotransmitter in the brain.

Similarities Between L and D Amino Acids:

L- and D-amino acids are two possible orientations of a particular amino acid in
nature. They are the mirror image of each other. Also, they can be considered as
the isomeric forms, stereoisomers or enantiomers. However, the simplest amino
acid, glycine, does not have stereoisomers. Both contain a carboxylic acid group, an
amine group, a carbon chain, and a hydrogen atom bound to the central carbon
atom of the amino acid. Further, this central carbon atom is called the alpha carbon
or the chiral carbon.

14 | P a g e
Difference Between L and D Amino Acids:

L-amino acid refers to a stereoisomer of a particular amino acid whose amino group
is on the left side in its Fisher projection while D-amino acid refers to the other
stereoisomer of the amino acid whose amino group is on the right side in its Fisher
projection. This explains the basic difference between L and D amino acids. Also,
while L-amino acids can rotate plane-polarized light counterclockwise in a process
called levorotation, D-amino acids can rotate plane-polarized light clockwise in a
process called dextrorotation. Another difference between L and D amino acids is
that L-amino acids have been superseded by R notation while the D-amino acids
have been superseded by S notation.

Moreover, L-amino acids are used by the cell to produce proteins while D-amino
acids occur in the cell wall of bacteria.

L-amino acids are a form of stereoisomers whose amine group occurs in the left
side of the Fisher projection. On the other hand, D-amino acids are the other form
of stereoisomers whose amine group occurs on the right side of the Fisher
projection. L-amino acids are a form of stereoisomer which is abundant in proteins.
This is the difference between L and D amino acids.

Properties of Amino Acids

- Each amino acid has both an acidic and basic group as you can see from its
structure. This is the reason they behave like salts.

- Any amino acid in the dry state is in crystalline form. They exist as a dipolar ion.
The COOH group exists as an anion. And the NH2 group exists as a cation. This
dipolar ion has a special name “Zwitter ions’.

- In aqueous solution, alpha amino acids exist in equilibrium between a cationic

form, an anionic form and dipolar ion.

15 | P a g e
- The Isoelectric point is the pH point at which the concentration of zwitter ions is
the highest ad the concentration of cationic and anionic form is equal. This point is
definite for every α-amino acid.

- They are generally water soluble and also have high melting points.
Physical Properties:

1. Amino acids are colorless, crystalline solid.

2. All amino acids have a high melting point greater than 200o

3. Solubility: They are soluble in water, slightly soluble in alcohol and dissolve with
difficulty in methanol, ethanol, and propanol. R-group of amino acids and pH of the
solvent play important role in solubility.

4. On heating to high temperatures, they decompose.

5. All amino acids (except glycine) are optically active.

6. Peptide bond formation: Amino acids can connect with a peptide bond involving
their amino and carboxylate groups. A covalent bond formed between the alpha-
amino group of one amino acid and an alpha-carboxyl group of other forming -CO-
NH-linkage. Peptide bonds are planar and partially ionic.

Chemical Properties:

1. Zwitterionic property

A zwitterion is a molecule with functional groups, of which at least one has a

positive and one has a negative electrical charge. The net charge of the entire
molecule is zero. Amino acids are the best-known examples of zwitterions. They
contain an amine group (basic) and a carboxylic group (acidic). The -NH2 group is
the stronger base, and so it picks up H+ from the -COOH group to leave a zwitterion.
The (neutral) zwitterion is the usual form amino acids exist in solution.

16 | P a g e
2. Amphoteric property

Amino acids are amphoteric in nature that is they act as both acids and base since
due to the two amine and carboxylic group present.

3. Ninhydrin test

When 1 ml of Ninhydrin solu-tion is added to a 1 ml protein solution and heated,

the formation of a violet color indicates the presence of α-amino acids.

4. Xanthoproteic test

The xanthoproteic test is performed for the detection of aromatic amino acids
(tyrosine, tryptophan, and phenylalanine) in a protein solution. The nitration of
benzoid radicals present in the amino acid chain occurs due to reaction with nitric
acid, giving the solution yellow coloration.

5. Reaction with Sanger’s reagent

Sanger’s reagent (1-fluoro-2, 4-dinitrobenzene) reacts with a free amino group in

the peptide chain in a mild alkaline medium under cold conditions.

6. Reaction with nitrous acid

Nitrous acid reacts with the amino group to liberate nitrogen and form the
corresponding hydroxyl.

17 | P a g e
Some common uses of amino acids
The industrial production of amino acids is an important worldwide business. The
first report of the commercial production of an amino acid was in 1908. It was then
that the flavoring agent monosodium glutamate (MSG) was prepared from a type
of large seaweed. This led to the commercial production of MSG, which is now
produced using a bacterial fermentation process with starch and molasses as
carbon sources. Glycine, cysteine, and D, L-alanine are also used as food additives,
and mixtures of amino acids serve as flavor enhancers in the food industry. The
amino acid balance of soy or corn protein for animal feed is significantly enhanced
upon the addition of the nutritionally limiting amino acids methionine and lysine.

Amino acids are used therapeutically for nutritional and pharmaceutical purposes.
For example, patients are often infused with amino acids to supply these nutrients
before and after surgical procedures. Treatments with single amino acids are part
of the medical approach to control certain disease states. Examples include L-
dihydroxyphenylalanine (L-dopa) for Parkinson disease; glutamine and histidine to
treat peptic ulcers; and arginine, citrulline, and ornithine to treat liver diseases.

Certain derivations of amino acids, especially of glutamate, are used as surfactants

in mild soaps and shampoos. D-Phenylglycine and D-hydroxyphenylglycine are
intermediates used for the chemical synthesis of β-lactam antibiotics (e.g.,
synthetic versions of penicillin). Aspartame is a sweetener prepared from the
individual component amino acids aspartic acid and phenylalanine.

18 | P a g e
Biomedical Importance of amino acids
In addition to supplying the monomer units by which the lengthy polypeptide
chains of proteins are synthesized, the L-α-amino acids as well as their derivatives
take part in cellular functions as various as neural transmission plus the
biosynthesis of porphyrins, purines, pyrimidines, and urea. Small polymers of
amino acids named peptides carry out dominant tasks in the neuroendocrine
system as hormones, hormone-releasing factors, neuromodulators, or
neurotransmitters. Amino acid supplements may help you recover faster, improve
performance and help build muscle. Additionally, supplementing with amino acids
can aid in the formation of hormones, enzymes, and antibodies. Although proteins
include only L-α-amino acids, micro-organisms intricate peptides that includes both
D- and L-α-amino acids. Numerous of these peptides are of therapeutic
importance, for example the antibiotics bacitracin and gramicidin A and the
antitumor agent bleomycin. A number of other microbial peptides are poisonous.
The cyanobacterial peptides microcystin and nodularin are fatal in big dosage
amounts, even though little amounts encourage the occurrence of hepatic tumors.
Not humans nor any other higher animals can synthesize 10 of the 20 common L-
α-amino acids in quantities sufficient to aid infant growth as well as keep health in
adults. Therefore, the human diet plan should consist of sufficient amounts of
those nutritionally necessary amino acids. We can also see the biomedical
importance in following category-

Anti-aging:

The amino acids arginine and carnitine forms creatine.

Natural skin functions are supported and the cells are stimulated to produce more
collagen and elastin.

Glutamine regulates the acid-base balance and firms the skin.

19 | P a g e
Arthritis and osteoporosis:

Methionine is known as an important organic sulfur donor.

Methionine is an important cartilage-forming substance.

Arginine supports the creation of bone.

Cholesterol and Diabetes:

Arginine lowers blood cholesterol level.

Arginine - an important amino acid for promoting the insulin absorption.

Fat-burning, Hair loss and Menopause:

Glutamine counteracts the storage of dietary fats and thus helps regulate weight.

Arginine boosts hair growth.

Arginine increases the elasticity of vessels and thereby helps with hot flushes.

Sleep, Mood and Performance:

The amino acid glutamine is involved in many metabolic processes. It stabilizes the
immune system, strengthens the intestinal cells and helps against stress,
depression and anxiety.

Commercial Importance of amino acids

Amino acids are used either alone or in combination, as flavor enhancers.
Monosodium glutamate is the most frequently used in food industry. Glycine and
alanine also enhance taste and flavor. Tryptophan, in association with histidine,
acts as an antioxidant to preserve milk powder. For the preservation of fruit juices,
cysteine serves as an antioxidant. Aspartame, a dipeptide (aspartyl-phenylalanine
methyl ester) produced by a combination of aspartic acid and phenylalanine, is

20 | P a g e
about 200 times sweeter than sucrose. It is used as a low-calorie artificial
sweetener in soft-drink industry.

There are certain essential amino acids that are deficient or limiting in plant
proteins. These include lysine, methionine, threonine and tryptophan. Addition of
the deficient amino acid(s) improves the nutritional quality of human foods as well
as animal feeds. Thus, bread enriched with lysine, soy products supplemented with
methionine are of better nutritional value. Methionine added soy bean meal is a
better feed for pigs and other animals.

The amino acids can be used as medicines. Essential amino acids are useful as
ingredients of infusion fluids, for administration to patients in post-operative
treatment.

Amino acids serve as starting materials for producing several compounds. Glycine
is used as a precursor for the synthesis of glyphosate (a herbicide), while threonine
is the starting material for the production of azthreonam (another herbicide). Poly-
methyl glutamate is utilized for manufacturing synthetic leather. Some amino acids
in the form of N-acyl derivatives are useful for the preparation of cosmetics.

Nullomers are codons that in theory code for an amino acid, however in nature
there is a selective bias against using this codon in favor of another, for example
bacteria prefer to use CGA instead of AGA to code for arginine. This creates some
sequences that do not appear in the genome. This characteristic can be taken
advantage of and used to create new selective cancer-fighting drugs and to prevent
cross-contamination of DNA samples from crime-scene investigations. Amino acids
are important as low-cost feedstock. These compounds are used in chiral pool
synthesis as enantiomerically pure building blocks.

Amino acids have been investigated as precursors chiral catalysts, such as for
asymmetric hydrogenation reactions, although no commercial applications exist.

21 | P a g e
Amino acids have been considered as components of biodegradable polymers,
which have applications as environmentally friendly packaging and in medicine in
drug delivery and the construction of prosthetic implants.[102] An interesting
example of such materials is polyaspartate, a water-soluble biodegradable polymer
that may have applications in disposable diapers and agriculture.[103] Due to its
solubility and ability to chelate metal ions, polyaspartate is also being used as a
biodegradable anti-scaling agent and a corrosion inhibitor.[104][105] In addition,
the aromatic amino acid tyrosine has been considered as a possible replacement
for phenols such as bisphenol A in the manufacture of polycarbonates.

Identification of amino acids

1. Solubility test

2. Ninhydrin test for α-L amino acids

3. Xanthoproteic test for Aromatic amino acids

4. Millon’s test for amino acids containig hydroxy

phenyl group

5. Sakaguchi Test

6. Detection of amino acids containing sulfhydral group (- SH)/

Lead Sulfite Test

1. Solubility test:

The physical proprieties of amino acid are mainly result of their structure, both the
solid state and in various solutions.

22 | P a g e
Objective:
Investigate the solubility of selected amino acid in various solutions.

Principle:
Polar amino acids are more soluble in water than non-polar, due to presence of
amino and carboxyl group which enables amino acids to accept and donate protons
to aqueous solution.

2. Ninhydrin test:

Objective:
To detect α-L-amino acids. Can be used also to detect free amino and carboxylic
acid groups on proteins and peptides. All amino acids that have a free amino group
will give positive result (purple color), while not free amino group-proline- will give
a (yellow color).

Principle:
-Ninhydrin (tri ketohydrindene hydrate) degrades a.a into aldehydes, ammonia and
CO2 (on pH range 4-8) though a series of reactions. The net result is ninhydrin in a
partially reduced from hydrindantin. Ninhydrin then condenses with ammonia and

hydrindantin to produce an intensely blue or purple pigment, sometimes called

ruhemann's purple.

The color varies slightly from acid to acid. Proline and hydroxy-proline (amino acids)
give yellow color. Many substances other than amino acids, such as amines will
yield a blue color with ninhydrin, particularly if reaction is carried out on filter
paper.

Ninhydrin is a strong oxidizing agent, it should be handled with care, and applied
apart from contact with skin or eyes, gloves and mask is a must, using hood is

23 | P a g e
required, if accidently get in touch with the skin, the resulting stains is a temporarily
one, that will be eliminated within 24 hours.

3. Xanthoproteic test:

Objective:
This test is used to differentiate between aromatic amino acids which give positive
results and other amino acids. Amino acids containing an aromatic nucleus form
yellow nitro derivatives on heating with concentrated HNO3. The salts of these
derivatives are orange in color.

Principle:
Conc. HNO3 reacts with the aromatic rings that are derivatives of benzene giving
the characteristic nitration reaction. Amino acids tyr. and typ. contain activated
benzene rings which are easily nitrated to yellow colored compounds. The aromatic
ring of phenyl alanine dose not react with nitric acid despite it contains a benzene
ring, but it is not activated, therefore it will not react.

4. Millon's test:

Objective:
This test is specific for tyrosine, the only amino acid containing a phenol group, a
hydroxyl group attached to benzene ring.

Note: All phenols (compound having benzene ring and OH attached to it) give
positive results in Millon’s test.

Principle:
The phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then
the nitrated tyrosine complexes mercury ions in the solution to form a brick-red

24 | P a g e
solution or precipitate of nitrated tyrosine, in all cases, appearance of red color is
positive test.

5. Sakaguchi Test:

Objective:
Sakaguchi test is a specific test for detection of amino acid containing gauanidium
group [R-NH-C= (NH2)2+ -NH2]. In other word it’s a test for guanidines, i.e. arginine.

Principle:
In alkaline solution, arginine reacts with α-naphthol and sodium hypobromite
/chlorite as an oxidize agent, to form red complexes as a positive result.

6. Detection of amino acids containing sulfhydral group (- SH)- Lead Sulfite Test:

Objective:
This test specific for–SH containing amino acid (Cysteine).

Principle:
- Some of sulfur in cystine, is converted to sodium sulfide by boiling with 40% NaOH.

- The Na2S can be detected by the precipitation of PbS from an alkaline solution.

- The amino acids containing sulfhydryl group when heated with base, the
sulfhydryl group and disulfhydryl are directly converted to inorganic sulfur. Which
is confirmed by the black precipitate of PbS (lead sulfide) when adding lead acetate
Pb(CH3COO)2.

25 | P a g e