What are amino acids? - PSAU · PDF fileAll amino acids found in proteins are of the L-configuration. However, D-amino acids are found in some ... amines left over from proteins sloughed

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-



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-)



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.





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.





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



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.



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



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.



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



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



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



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



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



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 &



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 )



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



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.



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

Documents

What are amino acids? - PSAU · PDF fileAll amino acids found in proteins are of the L-configuration. However, D-amino acids are found in some ... amines left over from proteins sloughed