Denaturation of Proteins

Introduction

Denaturation of proteins involves the disruption and possible destruction of both the secondary
and tertiary structures. Since denaturation reactions are not strong enough to break the
peptide bonds, the primary structure (sequence of amino acids) remains the same after a
denaturation process. Denaturation disrupts the normal alpha-helix and beta sheets in a
protein and uncoils it into a random shape.

Denaturation occurs because the bonding interactions responsible for the secondary structure
(hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are
four types of bonding interactions between "side chains" including: hydrogen bonding, salt
bridges, disulfide bonds, and non-polar hydrophobic interactions. which may be disrupted.
Therefore, a variety of reagents and conditions can cause denaturation. The most common
observation in the denaturation process is the precipitation or coagulation of the protein.

Denaturation of certain globular proteins is accompanied by an increase in the relative viscosity

of their solutions and a decrease in their diffusion constant. These effects are indicative of
changes in molecular dimensions and can be used for studying the denaturation of a protein
and its apparent reversal.

Conclusion

Protein stability is a critical factor in protein drug formulation, drug efficacy, and storage shelf
life. When proteins become unstable, molecules unfold and aggregate, causing the viscosity of
the protein to increase. Aggregated proteins can cause an immune response that leads to
serious side effects. This makes viscosity a key ingredient in determining protein stability, an
integral part of protein formulation.

Protein stability depends on temperature, pH, concentration and many other factors. For
example, as the temperature increases, protein unfolds and loses tertiary and secondary
structures. As unfolding progresses, proteins begin to aggregate, which could lead to the
precipitation or formation of a gel-like structure.

Measuring viscosity is one technique to monitor the tertiary structure of a protein. A folded
protein is a relatively compact body. In the denatured protein the peptide chain is unfolded into
many different random arrangements, each of these have more interactions with the solvent
than does the folded protein and so the unfolded protein drags more solvent around with it as
it moves. Its effective average size is increased. Also, depending on concentration, unfolded
chains can interact (tangle) with one another and so rigidify a solution.
The viscosity of a protein solution is increased by the denaturation of the protein. This is true
both when there is the formation of protein aggregates which occlude water and when there is
no aggregation.

Determination of Saponification and Iodine Numbers of Some Lipids

Introduction

Fatty acids, which are carboxylic acids, are composed of long hydrocarbon chain and a terminal
carboxyl group. Under physiological conditions, the carboxyl group is normally ionized. Fatty
acids can either be saturated, where all carbon-carbon bonds are single bonds, or unsaturated,
where one or more double bonds are observed in the hydrocarbon chain. Saturated fatty acids
are usually observed to be solid at room temperature. In contrast, unsaturated fatty acids tend
to be liquid due to the double bonds that cause the hydrocarbon chain to bend; hence, allowing
the free movement of the molecule. On the other hand, fats and oils are derivatives of fatty
acids,
and in many organisms, they are stored forms of energy, such as triacylglycerol or triglycerides.

Triglycerides are a major energy reserve and the principal neutral derivatives of glycerol found
in animals. It consists of a glycerol esterified with three fatty acids. The ester linkages between
the polar hydroxyls of glycerol and the polar carboxylates of the fatty acids result into the non-
polar and hydrophobic nature of triglycerides. The hydrolysis of fats or oils under basic
conditions, such that glycerol and the salt of the corresponding fatty acid are obtained, is called
saponification. An application of saponification in the industry is the production of soaps. In
fact, saponification literally means, “soap making”. In the industry, it is important to know the
amount of free fatty acid that is present since the refining loss is dependent on it. This can be
done by detemining the amount of alkali that can neutralize the fatty acids. In here, a known
amount of the fat is heated with a strong aqueous caustic soda solution, which then cause the
conversion of free fatty acids into soap; followed by removal of soap. The amount of fats that
have remained is then subtracted from the original amount of the fats that have been used for
the test, which gives the estimated loss.

The number of milligrams of alkali, which is commonly potassium hydroxide, needed to

neutralize the fatty acid is called the saponification number. This helps in characterizing the
nature of the fatty acids of the fat. Moreover, it also corresponds to the average molecular
weight of all the fatty acids present. Lower saponification number is expected from long chain
of fatty acids, due to a relatively fewer number of carboxylic functional groups per unit mass of
the fat.

Furthermore, unsaturated fatty acids can be converted to saturated acid through the process
hydrogenation. In this process, oxygen or other halogens can combine with the unsaturated
fatty acids, depending on the degree of unsaturation, to convert it to saturated ones. One way
to estimate the unsaturation of fats is to determine its iodine value. This value corresponds to
the amount of grams of iodine consumed by 100g of fat. The degree of unsaturation is directly
proportional to the iodine value. In this experiment, the students are to determine the
saponification and iodine number of sample oils using standard protocols.

Conclusion

Lipids can be classified as: saponifiable lipids and unsaponifiable lipids. Saponifiable lipids are
open-chain compounds containing polar heads and long nonpolar chains. Among the
saponifiable lipids are triacylglycerols, waxes, sphingolipids and phospholipids. Unsaponifiable
lipids on the other hand are those with fused rung compounds such as steroid and cholesterol.

On refluxing with alkali, triacylglycerols (fatty acid esters) are hydrolyzed to give glycerol and
potassium salts of fatty acids (soap). Such process is known as, Saponification. Refluxing is a
necessary process in finding the saponification value because reflux is a technique involving the
condensation of vapors and the return of this condensate to the system from which it
originated.

Saponification is a process that produces soap, usually from fats and lye. In technical terms,
saponification involves base (usually NaOH) hydrolysis of triglycerides, which are esters of fatty
acids, to form the sodium salt of a carboxylates. In addition to soap, such traditional
saponification processes produces glycerol. "Saponifiable substances" are those that can be
converted into soap. To remove fat droplets and it also indicates that saponification is
complete.

The saponification value is the number of milligrams of KOH required to neutralize the fatty
acids resulting from the complete hydrolysis of 1g of fat. The saponification value gives an
indication of the nature of the fatty acids constituent of fat and thus, depends on the the
average molecular weight of the fatty acids constituent of fat. The greater the molecular weight
(the longer the carbon chain), the smaller the number of fatty acids is liberated per gram of fat
hydrolyzed and therefore, the smaller the saponification number and vice versa.