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In this article we will discuss about the instability and stability aspects of polypeptides and proteins.
I. Instability Aspects of Polypeptides and Proteins:
i. Physical Instability:
Physical instability in peptides and proteins can be manifested as denaturation, absorption, aggregation and precipitation.
a. Denaturation:
Peptides and polypeptides are made up of both polar and non-polar amino acids. When such peptide come in contact with aqueous environment, the amino acid fold up on themselves to form a globular molecule in which hydrophilic amino acids are exposed to the aqueous medium.
If these molecules are then placed in hydrophobic environment (non-aqueous solvent) for purification or formulation, they will unfold, leading to the exposure of hydrophobic amino acids to the environment.
The rearrangements results in loss of tertiary and quaternary structure. This can also occur if solvent is changed from aqueous to mixed solvent is changed from aqueous to mixed solvent (example: alcohol-water, acetone water). Changes in pH also effect the structure of proteins, since this alter the ionisation of carboxylic and amino groups.
Similarly, change in ionic strength affects the charge carried by the molecules and lead to denaturation Changes in temperature also cause unfolding of polypeptides and proteins. Increase in temperature breaks the hydrogen bonds and hence, there will be loss of secondary or tertiary structure.
b. Absorption:
Proteins and peptides contain both polar and non-polar amino acids residues (amphillic). Thus, like other surfactants, they also have a tendency to be absorbed at interface, such as air-water, and air-solid.
The hydrophobic, non-polar amino acids prefer hydrophobic environment such as air, surfaces of glass or plastic. Thus, peptides and protein may rearrange and denature on absorption at an interface. Once absorbed, they form hydrophobic or van der Waals or ion-pair bonds with the surface.
This may lead to further denaturation. The absorption can occur during storage, processing, formulation, or delivery. Insulin is absorbed to the surface of delivery pumps, to glass and plastic containers and to plastic intravenous bags and tubing. Biological activity is changed or loss due to absorption. This also causes a reduction in the concentration of drug available.
c. Aggregation and Precipitation:
Denatured, unfolded protein may rearrange to form aggregates in which hydrophobic amino acid reduces of different molecules are associated together. More amount of aggregation leads to precipitation. Interfacial absorption leads to precipitation and aggregation.
The process is enhanced by the presence of hydrophobic surfaces. Presence of large air-water interface can accelerate precipitation. Agitation of protein and polypeptides can also lead to aggregation. This is due to introduction of air-bubbles as well as increased thermal motion of the molecules.
ii. Chemical Instability:
There is different reaction of proteins and polypeptides, which leads to chemical instability.
There are listed below:
a. Deamidation:
It involves hydrolysis of the side chain of amide linkage of an amino acid reduce to form a carboxylic acid. Some common proteins, which undergo in vitro deamidation, are human growth hormone insulin and prolactin. Glutamine and asparagines are the amino acid, which undergo deamidation.
The rate of deamidation is increased by an increase in pH, temperature and ionic strength. The tertiary strength of protein resists deamidation, For example, tertiary structure of trypsin prevents deamidation. Deamidation reduces biological activity. For example, the activity of ACTH (adrenal corticotrophin hormone) is reduced by deamidation.
b. Oxidation:
Oxidation occurs in the side chains of histidine, methionine, lysine, tyrosine and tryptophan resides in protein. It is commonly during synthesis, isolation and storage. Atmospheric oxygen oxidises methionine under acidic conditions. Oxidising agents like hydrogen peroxide, iodine and dimethyl sulphoxide are also responsible for oxidation.
Oxidations are reduced biological activity. For example, oxidation of methionine in gastrin, and corticotropin results in loss of activity. In some proteins such as Lysozyme, biological activity can be restored by reduction. Some other proteins such as glucagons can retain the activity even after oxidation.
c. Racemisation:
Except glycine, all other amino acids are chiral at carbon bearing the side chain and hence are susceptible to Racemisation. Thus racemisation may convert the protein non-metabolisable because the racemic peptide bonds are inaccessible to proteolytic enzymes and can reduce biological activity.
d. Disulphide Exchange:
Breaking and incorrect reformation of disulphide bonds may alter the three-dimensional structure of a protein and hence can change or alter biological activity. The reaction is catalysed by thiols, which arise as a result of hydrolytic cleavage of disulphides.
e. Proteolysis:
Proteolysis is the cleavage of protein molecule by the breakage of peptide bond. This depends on the residues involved. Asparagine residue, particularly the bond between asparagines and proline is highly susceptible to cleavage. But the peptide bonds, in general, are stable at neutral pH and room temperature. Proteolysis occurs upon heating. For example; heating at 90-100°C inactivates lysozyme.
II. Stability Aspects of Polypeptides and Proteins:
i. Physical Stability:
Certain additives given in table below can be used to improve physical stability of polypeptides and proteins. Specific ion-binding sites introduced on the protein molecules leads to improve stability. Both nonionic and iconic surfactants can stabilise proteins by preventing their absorption at the interface. They also inhibit aggregation and precipitation Salts can decrease denaturation and increase the stability by nonspecific or specific ion binding to the protein.
At low concentration in aqueous solution, polyhydric alcohol such as glycerol can stabilise proteins by selective salvation. Here water molecules pack around the protein to exclude the hydrophobic alcohol molecules and increase stability. The reverse occurs at high concentration of the polyalcohol.
ii. Chemical Stability:
Different methods are used to improve chemical stability.
They are described below:
Modification of Protein Molecules:
Synthetic polymers like PEG and polyoxyethylene can be coupled to protein molecules chemically to enhanced stability. However, there are chances of reduction or complete loss of biological activity. Comparatively, lipids are better coupling agents. Lipid coupled with insulin can increase the absorption of insulin and hence increase the activity.
Site Directed Mutagenesis:
These techniques created specific mutations that alter the amino acids sequence of proteins and create new proteins. The process can be designed to improve both stability as well as specificity of proteins.
Here, specific amino acids, which are reactive, can be replaced with others, which are non-reactive, while retaining the biological activity of the protein. It is also possible to introduce specific nonbinding sites to improve the stability. Disulphide bonds can be introduced, which can stabilise the original confirmation and provide stability against denaturation.
Choice of Conditions:
Chemical stability can be obtained by appropriate choice of conditions, such as physical state, pH temperature, preservatives and ionic strength Peptides and proteins can be stabilised if they are in dry state. Lower temperature also enhanced stability.
Dosage forms can be formulated in a way that they are solid and freezedried products. At the time of administration, they can be reconstituted. The stability of proteins and polypeptides is an important consideration in the selection of route of administration and appropriate dosage form.