Protein Denaturation

Denaturation is a process that changes the molecular structure without breaking any of the
peptide bonds of a protein. It is that involves transformation of a well-defined, folded structure of a
protein to an unfolded state. Protein denaturation is therefore the change in the secondary, tertiary,
and quaternary structure of a protein. There is no change in the primary structure. In other words,
denaturation does not involve breaking of peptide bonds. Protein denaturation may occur as a result
of heat, pH, change in salt concentration or high mechanical shear (whipping, shaking).

Any of these factors may cause breaking of hydrogen bonds and salt bridges. As a result, the
protein unfolds and side chains that were buried in the center of the molecule become exposed. They
are then available to react with other chemical groups, and in most cases the denatured protein
precipitates. Protein denaturation
generally is not reversible.
The proteins of egg white are readily denatured by heat and by surface forces when egg white
is whipped to a foam. Meat proteins are denatured in the temperature range 57–75 °C, which has a
profound effect on texture, water holding capacity, and shrinkage. Foods may be denatured, and their
proteins destabilized, during freezing and frozen storage. Fish proteins are particularly susceptible to
destabilization. After freezing, fish may become tough and rubbery and lose moisture. The caseinate
micelles of milk, which are quite stable to heat, may be destabilized by freezing. On frozen storage
of milk, the stability of the caseinate progressively decreases, and this may lead to complete
coagulation
Denaturation usually involves loss of biological activity and significant changes in some
physical or functional properties such as solubility. Enzymes are inactivated and so the reactions that
they catalyzed no longer can take place. The destruction of enzyme activity by heat is an important
operation in food processing.
Denaturation may be desirable and can be deliberately brought about by food processing.For
example adding acid to milk to form cottage cheese, or inactivating enzymes by heat, as occurs when
vegetables are blanched before freezing. Blanching is a mild heat treatment that denatures and
inactivates enzymes that would cause rancidity or discoloration during frozen storage. On the other
hand, in general, denatured proteins are more digestible than native proteins.
Factors influencing protein denaturation
1. Temperature- Heat is the most commonly used denaturing agent in foods. Proteins undergo
varying degree of denaturation during processing, depending on time and temperature
employed and this can affect their functional properties in foods. When a protein solution is
gradually heated above a critical temperature, it undergoes a sharp transition from the native
state to the denatured state.
The temperature-induced denaturation of proteins is primarily due to the effect of temperature
on the stability of noncovalent interactions. Hydrogen bonding and electrostatic interactions,
which are exothermic in nature, are destabilized, and hydrophobic interactions, which are
endothermic, are stabilized as the temperature is increased. The strength of hydrophobic
interactions reaches a maximum at about 70°C–80°C and decreases thereafter.
2. Hydrostatic pressure- Most proteins undergo pressure-induced denaturation in the range of
1–12 kbar at 25° C as evidenced from changes in their spectral properties. The midpoint of
pressure-induced transition occurs at 4–8 kbar. Pressure-induced denaturation occurs mainly
because proteins are flexible and compressible. Pressure-induced protein denaturation is
highly reversible. Most enzymes, in dilute solutions regain their activity once the pressure is
decreased to atmospheric pressure. Pressure processing, unlike thermal processing, does not
harm essential amino acids, natural color, and flavor, nor does it cause toxic compounds to
develop. Thus, processing of foods with high hydrostatic pressure, though may be costly, may
prove to be advantageous for certain food products.
3. Shear- High mechanical shear generated by shaking, kneading, whipping, etc., can cause
denaturation of proteins. Many proteins denature and precipitate when they are vigorously
agitated. In this case, denaturation occurs because of incorporation of air bubbles and
adsorption of protein molecules to the air–liquid interface. Since the air–liquid interface has
an excess free energy compared to the bulk phase, proteins undergo conformational change at
the interface. The extent of conformational change depends on the flexibility of the protein.
Highly flexible proteins denature more readily at an air–liquid interface than do rigid proteins.
Upon interfacial denaturation, the nonpolar residues of denatured protein orient toward the
gas phase and the polar residues orient toward the aqueous phase.
4. pH- Proteins are more stable against denaturation at their isoelectric point (neutral pH) than
at any other pH. At neutral pH, most proteins are negatively charged, and a few are positively
charged. However, when the pH is shifted to very low or very high values, the net charge of
the protein changes accordingly and strong intramolecular electrostatic repulsion causes
swelling and unfolding of the protein molecule. The degree of unfolding is greater at extreme
alkaline pH values than it is at extreme acid pH values. Denaturation caused by high alkaline
 pH is attributed to ionization of partially buried carboxyl, phenolic, and sulfhydryl groups that
cause unraveling of the polypeptide chain as they attempt to migrate to the aqueous
environment. The pH-induced denaturation is mostly reversible.
5. Organic solvent- the net effect of an organic solvent on protein structure usually depends on
the magnitude of its effect on various polar and nonpolar interactions. At low concentration,
some organic solvents can stabilize enzymes against denaturation. At high concentrations,
however, all organic solvents cause denaturation of proteins because of their solubilizing
effect on nonpolar side chains.
6. Organic solutes-Organic solutes, notably urea and guanidine hydrochloride (GuHCl), cause
denaturation of proteins. For many globular proteins the midpoint of transition from the native
to denatured state occurs at 4–6 M urea and at 3–4M GuHCl at room temperature. Complete
denaturation often occurs in 8 M urea and in about 6 M GuHCl. GuHCl is a more powerful
denaturant than urea because of its ionic character.
7. Chaotropic salts- Salts affect protein stability in two different ways. At low concentrations,
ions interact with proteins via nonspecifi¬c electrostatic interactions. This electrostatic
neutralization of protein charges usually stabilizes protein structure. Complete charge
neutralization by ions occurs at or below 0.2M ionic strength and it is independent of the
nature of the salt. However, at higher concentration (>1 M), salts have ion specific effects that
influence the structural stability of proteins. Salts such as Na2SO4 and NaF enhance, whereas
NaSCN and NaClO4 weaken, protein stability. Protein structure is influenced more by anions
than by cations.
Nutritional properties of proteins
Proteins differ in their nutritive value. Several factor such as essential amino acids content
and digestibility, contribute to these differences. The daily protein requirement therefore
depends on the type and composition of proteins in a diet.
 Protein quality
The ‘quality’ of protein is related mainly to its essential amino acids content and
digestibility. High-quality proteins are those that contain all the essential amino acids
at levels greater than the FAO/WHO/UNU reference levels, and a digestibility
comparable to or better than those of eggs white or milk proteins. Animal proteins are
better ‘quality’ than plant proteins. Proteins of major cereals and legumes are often
deficient in at least one of the essential amino acids. While proteins of cereals, such as
rice, wheat, barley and maize, are very low in lysine and rich in methionine, those of
legumes and oilseeds are deficient in methionine and rich or adequate in lysine.
 Digestibility
Although the content of essential amino acid is the primary indicator of protein quality,
true quality also depends on the extent to which these amino acids are utilized in the
body. Thus, digestibility (bioavailability) of amino acids can affect the quality of
proteins. Food proteins of animal origin are more completely digested than those from
plant origin. Several factors affect digestibility of proteins.
a) Protein conformation-the structural state of a protein influences its
hydrolysis by proteases. Native proteins are generally less completely
hydrolysed then partially denatured ones. For example, treatment of
phaseolin (a protein from kidney beans) with a mixture of proteases
results only in limited cleavage of the protein resulting in liberation of
a 22,000 Da polypeptide as the main product. When heat-denatured
phaseolin is treated under similar conditions, it is completely
hydrolyzed to amino acids and dipeptides. Generally, insoluble fibrous
proteins and extensively denatured globular proteins are difficult to
hydrolyse.
b) Antinutritional factors- most plant protein isolates and concentrates
contain trypsin and chymotrypsin inhibitors and lectins. These
inhibitors impair complete hydrolysis of legumes and oilseed proteins
by pancreatic proteases. Lectins, which are glycoproteins, bind to
intestinal mucosa cells and interfere with absorption of amino acids.
Plant proteins also contains other antinutritional factors, such as tannins
and phytate. Tannins, which are condensation products of polyphenols,
covalently react with ε-amino groups of lysine residues. This inhibits
trypsin-catalysed cleavages of the polypeptides at lysine sites.
c) Processing- interaction of proteins with polysaccharides and dietary
fibre also reduces the rate and completeness of hydrolysis. This is
particularly important in extruded food products where high
temperature is often used. Protein undergo several chemical alterations
involving lysine residues when exposed to high temperatures and
alkaline pH. Such alterations reduce their digestibility. Reaction of
reducing sugars with ε-amino acids groups also decreases digestibility
of lysine.