Denaturation of Proteins

Koushik DasM.Sc. 1st sem

School of Industrial Fisheries Cochin University of Science &

Technology

Denaturation of Proteins

Can be defined as any change that alters the unique 3D conformation of a protein

molecule without causing concomitant cleavage of the peptide bonds

It leads to temporary or permanent loss of activity.

Denaturation is a process that disrupts secondary, tertiary, and quaternary structures.

The primary structure is not destroyed during denaturation.

∗ Denaturation∗ Any physical or chemical agent that destroys the

conformation of a protein is said to “denature” it.

∗ Examples:∗ Heat (boil an egg) Frying an egg is an example of

denaturation by heat. Heat disrupts intermolecular forces such as hydrogen bonding, and other polar interactions.

∗ Addition of 6M Urea (breaks H bonds)∗ Detergents (surface-active agents)∗ Reducing agents (break -S-S- bonds)∗ Acids/Bases/Salts (affect salt bridges)∗ Heavy metal ions (Hg2+, Pb2+)

Other denaturing agents:• Changes in pH, which alters the ability of the acidic and

basic side chains to form salt bridges• Organic compounds, which will disrupt the disulfide

bonds• Heavy metals that disrupt salt bridges and disulfide bonds• Mechanical agitation, which disrupts hydrogen bonds

Denaturation of Proteins

A protein will lose its biological activity if it loses its three-dimensional shape.

Denaturation of Proteins

© 2011 Pearson Education, Inc.Chapter 9 7

Denaturation of Proteins

∗ At Very High or Low pH.

∗ At Very High Temperatures.

∗ By Heavy Metal Ions.

∗ By Small Polar Molecules.

When and How are Proteins Denatured?

∗ Affects Ionic Bonds.

∗ A High (>9) or Low pH (<3) will neutralise the charge on one of the ionically bonded ions.

∗ Hence the ionic bond is broken.

Denaturation at HIGH or LOW pH

∗ As the temperature increases the energy of the protein increases.

∗ As the energy increases bonds are broken in the following order:

Denaturation at High Temperatures

• Van der Waal’sVan der Waal’s• Hydrogen BondsHydrogen Bonds• Ionic BondsIonic Bonds

∗ Certain metal ions will disrupt the Van der Waals’ forces in proteins.

∗ Heavy Metal Ions, such as Ag+ and Hg2+, also inhibit enzyme activity.

∗ They do so by reducing the SH groups in cysteine.

Denaturation by Metal Ions

PLEASE NOTE: Spelling mistake on pictures: --- The S-S bond is a DISULPHIDE bond.

∗ Urea ( CO(NH2)2 ) in concentrated solution will denature proteins.

∗ It disrupts the Hydrogen Bonds.

∗ This causes complete denaturation.

Denaturation by Small Polar Molecules

Denaturation at the Quaternary Level

- Dissociation of the subunits- Dissociation is usually accomplish by disrupting ionic

interaction between certain groups

- Urea, guanidine hydrochloride or dodecyl sulfate (detergent)

Denaturation at the Tertiary Level

- Heat and ultraviolet radiation- Extremes of pH (acidic and basic reagents)

- Ions of heavy metals (e.g. Hg2+, Ag+, Pb2+) forms mercaptides with thiol groups

- Organic solvents or non-polar additives (e.g. ethyl alcohol, formaldehyde and rubbing alcohol)

Denaturation at the Tertiary Level

- Urea and guanidinium chloride- Amphipaths can destabilize polar and non-

polar interactions- Polyethylene glycol – can destabilize

hydrogen bonds- Performic acid – can cause oxidation of

disulfide bonds- Mercaptoethanol –can cause disruption of

disulfide bonds

Denaturation of the Secondary Level

High temperature - will increase the vibration of atoms along hydrogen bonds

Denaturation at the Primary Level

- Hydrolysis- Enzymes (peptidases)- Cyanogen bromide, N-

bromosuccinimide

Why is Denaturation Sudden?

Concentration of denaturant or temperature

100%

0%

Nat

ive

Str

uct

ure

Critical value

COOPERATIVE PROCESS

Partly denatured structure is weaker so begins to change faster

∗ Some denaturation is reversible∗ Urea (6M) then add to H2O

∗ Some is irreversible∗ Hard boiling an egg

Denaturation

∗ Denaturation

Denaturation

Unfolding and Renaturation (refolding)

Anfinsen’s Experiment: Spontaneous folding of denatured ribonuclease

The first proof for “sequence determines structure”

∗ Curling straight hair or straightening curly hair requires protein denaturation.

∗ Both processes require disruption of disulfide bonds found in the hair protein keratin.

∗ The disruption of disulfide bonds reshapes the hair and forces the reformation of disulfide bonds in new places.

.

Cont..

Ammonium thioglycolate, which reduces (breaks) disulfide bonds, and hydrogen peroxide (reforms disulfide bonds) are two chemical agents used in hairstyling.

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Denaturation of Proteins

∗ The process of denaturation is used as an antidote for lead or mercury poisoning.

∗ Egg whites can be given to an individual who has ingested a heavy metal. Egg whites are denaturated by the heavy metals and a precipitate is formed.

∗ Vomiting is induced to eliminate the metal-protein precipitate.

Denaturation of Proteins

Behavior of Denatured ProteinHydrophobic core

Hydrophilic surface

NATIVE

AGGREGATEDor other ingredient interactions

DENATURED

Unfolding forces some hydrophobic AA to surface

Fast under non-physiological conditions

Slow under physiological conditions

∗ Loss of enzymatic activity (death)/ Loss of biological activity

∗ Destruction of toxins∗ Improved digestibility∗ Loss of solubility∗ Changes in texture∗ Altered water binding capacity∗ Increased intrinsic viscosity∗ Inability to crystallize

Consequences of Denaturation

∗ reduce the protein to a simple polypeptide chain∗ slow freezing and variability of storage conditions, cause this

denaturation. ∗ A denatured protein has not only lost its ability to function as an

enzyme, but also its "water-holding“ ability.∗ This results in denatured fish flesh dripping excessively when thawed (a

situation known as "drip-thaw"), and appearing white, dull and spongy, and upon chewing becoming

fibrous and tasteless.

Protein Denaturation- chemical change during fish autolysis

∗ The conversion of a biologically functional molecule into a non-functional form

∗ There are many denatured states but one native state∗ Proteins can regenerate to their native state but

slowly∗ Denatured proteins have a greater tendency to

aggregate.

Denaturation