PROPERTIES OF PROTEIN

DenaturationRefers to any disruption in the

secondary, tertiary or quaternary levels of protein structure.

It does not cleave the peptide bonds, therefore the primary level of structure is not altered.

(+) precipitation or coagulationMost significant consequences of

denaturation – loss of biological activity

Reaction withHeatGentle heating causes reversible

denaturation of proteinVigorous heating denatures protein

irreversibly by disrupting several types of bonds.

Sample Observation

Albumin (+) formation of white coagulate

Gelatin (+) formation of colorless coagulate

Casein (+) formation of white coagulate

Reaction withHeatEgg white, contains high percentage

of protein, coagulates on heating Heat coagulates and destroys protein

present in bacteria – thus, sterilization of instruments and clothing use in operating rooms requires the use of high temperatures.

Reaction withSalts of Heavy MetalsHeavy metal salts, such as mercuric

chloride (bichloride of mercury) or silver nitrate (lunar caustic), lead, precipitate protein. It also cleaves –SH bonds.

Denatured protein irreversibly by disrupting the salt bridges and the disulfide bonds present in the protein.

Poisonous if taken internally because they coagulate and destroy protein present in the body.

Reaction withSalts of Heavy MetalsEgg White - Antidote for mercuric

chloride or silver nitrate when these poisons are taken internally and is precipitate out.◦ Egg white colloid has a charge opposite to

that of the heavy metal ion and so attracts it.Sample Reagent Observation

Albumin + AgNO3(+) formation of white

coagulate - more

Albumin + Hg2Cl2 (+) formation of white coagulate - less

Reaction withAlkaloidal ReagentsAlkaloidal reagents, such as tannic

acid and picric acid form insoluble compounds with proteins.

Alkaloidal reagents denature protein irreversibly by disrupting salt bridges and hydrogen bond.

Sample Reagent ObservationAlbumin + sat. picric acid Formation of yellow coagulate

Albumin + tannic acid Formation of flesh coagulate

Albumin + TCA Formation of milky white coagulate

Reaction withAlkaloidal ReagentTannic acid has been used extensively

in the treatment of burns.◦ When applied to a burn area, it causes the

protein to precipitate as a tough covering, thus reducing the amount of water loss from the area and reduces exposure to air.

Reaction withConcentrated AcidsChanges in pH can disrupt hydrogen

bonds and salt bridges, causing irreversible denaturation.

Proteins are coagulated by strong acids as concentrated HCl, sulfuric and nitric acids.

Sample Reagent ObservationAlbumin + HNO3 Formation of yellow

coagulate

+ excess HNO3

Yellow coagulate disappears

Albumin + HCl Formation of white coagulate

+excess HCl White coagulate disappears

Reaction withConcentrated AcidsHeller’s Ring test

◦ Used to detect the presence of albumin in urine

◦ A layer of concentrated nitric acid is carefully placed under a sample of urine in a test tube.

◦ If albumin is present, it will precipitate out as a white ring at the interface of the two liquids.

◦ If acid or base remains in contact with protein for a long period of time, the peptide bond will break.

Reaction withAlcoholAlcohol coagulates (precipitates) all

types of proteins except prolamines.Alcohol denatures protein by forming

hydrogen bonds that compete with the naturally occurring hydrogen bonds in the proteins.

Process is not reversible70% alcohol – used to disinfect

because of its ability to coagulate the proteins present in the bacteria

Reaction withAlcohol(+) formation of white coagulate

Sample Reagent Observation

Albumin + 95% ethyl alcohol

Greatest amount of white

coagulateAlbumin + 70% ethyl

alcoholModerate

amount of white coagulate

Albumin + 40% ethyl alcohol

Least amount of white coagulate

TEST FOR PROTEINS & AMINO ACID

BIURET TESTSolution contents:

◦KOH, CuSO4, Potassium sodium tartarate

Test for:◦Peptide bonds. (Proteins and peptones

give positive results)Theory:

◦Following compound is formed between the -C-N-C-back bone of the proteins and the copper(II) ions in the solution which results in a violet colour complex.

BIURET TEST

NINHYDRIN TESTSolution content:

◦NinhydrinTest for:

◦Amine group (mainly primary amine groups in proteins, peptones and amino acids)

Theory:◦When reacting with these free amines,

a deep blue or purple color known as Ruhemann's purple is produced.

NINHYDRIN TESTRuhemann’s Purple

◦A blue-violet dye formed in the reaction of ninhydrin with amino acids

◦ Any primary (C-terminal) amino acid will undergo the Ist reaction and will produce a blue colour

◦ Secondary (N-terminal ) amino group containing amino acid, proline will give a yellow/orange colour (II).

NINHYDRIN TEST

XANTHOPROTEIC TESTSolution contents:

◦Concentrated HNO3

Test for:◦Activated benzene rings (tyrosine and

tryptophan)Theory:

◦The aromatic groups in the amino acids can undergo nitration with nitric acid and give in yellow coloured products if the benzene ring is activated.

XANTHOPROTEIC TESTPhenylalanine, though a derivative of

benzene doesn’t undergo nitration because it’s not activated like tryptophan and tyrosine.

If you accidentally spill any nitric acid you may experience yellow stains because of this xanthoproteic reaction because the epidermal cells of the skin may contain proteins with aromatic amino acids.

XANTHOPROTEIC TEST

HOPKIN’S COLE TESTSolution content:

◦Sodium nitroprusside, NH3(aq)

Test for:◦Indol group. (Tryptophan)

Theory:◦The indol group reacts with

glycoxylic acid in conc. Sulfuric acid to form a purple colour / ring.

HOPKIN’S COLE TEST

MILLON’S TESTSolution contents:

◦Mercury (Hg22+, Hg2+) in nitric acid

Test for:◦Phenol groups (Tyrosine)

Theory: ◦Firstly the phenol ring is nitrated with

conc. nitric acid and the nitrated product will react with the mercury ions in the solution to give in either a deep red color solution or precipitate.

MILLON’S TEST [Hg2+](Some tyrosine containing

proteins will give in a white precipitate [Hg2

2+] which would turn red when heated.

MILLON’S TEST

TEST FOR SULFUR GROUPSolution contents:

◦40% NaOH, Lead AcetateTest for:

◦Sulfur. (Cysteine, Methionine)Theory:

◦By boiling with NaOH, Sulfur in the amino acid is converted into NaS, which then precipitates as black PbS with the addition of lead acetate.

TEST FOR SULFUR GROUP(+) Black ppt