AN ASSIGNMENT ON:

DENATURATION OF PROTEINS

COURSE NAME:BIOCHEMISTRY AND CELLULAR BIOLOGY

COURSE CODE:PHARM 217

SUBMITTED TO:

SUBMITTED BY:

SL.NO NAME REGISTARTION NO1 RACHANA SARKAR 121030222 SUSMITA GHOSH 121030233 FURHATUN-NOOR 121030484 PRIYANKA FLORINA

KARMOKAR12103050

5 TAJRIAN RAHMAN 12103059

DATE OF SUBMISSION:

3RD MAY, 2013

UNIVERSITY OF ASIA PACIFIC

Biochemistry and Cellular Biology Page 1

CONTENT:

SL. NO : TOPICS: PAGE NO:1. Denaturation of Proteins 3

2. Examples of Denaturing of Proteins 4

3. Reversibility and Irreversibility of Denaturation Process

4

4. Mechanism of Denaturation of Proteins 4-5

5. The Causes of Protein Denaturation 6

6. Denaturing Agents Change in Temperature Changes in pH Heavy metal salts Reducing agents Detergents Agitation Alcohols Acid and Bases

7-10

7. How Denaturation Occurs at Levels of Protein Structures

Primary structure Secondary structure Tertiary structure Quaternary structure

10-11

8. Measuring of Protein Denaturation Loss of solubility Increased proteolysis Loss of biological activities Tritium-Hydrogen exchange Spectroscopic procedures

12-14

9. Denaturation at Interfaces 15

10. Benefits of Denatured proteins 16

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Denaturation of Proteins:

Denaturation of proteins:

Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), or heat.

If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation.

In simple terms, protein denaturation is break down of peptide bonds between the amino acids.

Fig: Organized molecular configuration is disturbed

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Example of Denaturing in Proteins:

A classic example of denaturing in proteins comes from egg whites, which are largely egg albumins in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking the thermally unstable whites turns them opaque, forming an interconnected solid mass. The same transformation can be effected with a denaturing chemical. Pouring egg whites into a beaker of acetone will also turn egg whites translucent and solid. Denaturing egg whites is irreversible.

The skin that forms on curdled milk is another common example of denatured protein.

Reversibility and irreversibility:

They are two types of denaturation of proteins: Reversible and irreversible.

In many proteins (unlike egg whites), denaturation is reversible (the proteins can regain their native state when the denaturing influence is removed). This was important historically, as it led to the notion that all the information needed for proteins to assume their native state was encoded in the primary structure of the protein, and hence in the DNA that codes for the protein.

Mechanism of denaturation of proteins:

Unfolding of mesophilic proteins occurs both at temperatures higher and lower than room temperature: the high temperature transition is generally referred to as “heat denaturation” whereas that at lower temperatures is known as “cold denaturation”. We have recently identified a protein, Yfh1, whose cold denaturation occurs at accessible temperatures close to 0°C and under physiological conditions at pH 7; that is, without the need to add denaturants. The first instance in which this system was used in a general sense to study the stability of proteins was a study on the influence of alcohols at low concentrations. Measuring both thermal denaturations, and hence the stability curve, in the presence of trifluoroethanol, ethanol and methanol, we

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observed an extended temperature range of protein stability. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. A second important application is illustrated by titin I28, the second case of a protein undergoing unbiased cold denaturation. The thermal stability of this protein cannot be determined by increasing the temperature because aggregation competes with unfolding.

The causes of protein denaturation:

1. Denaturation occurs when a protein is exposed to an extreme environment, such as a high level of salt, high temperature, and/or high level of acidity. Because of these extreme conditions, the function of the protein alters due to deformities along their bonds and can be permanent or temporary based on several factors such as duration of exposure or exactly how "extreme" an extreme condition was. For example, some proteins are able to withstand exposure to extreme heat for several minutes and still retain original function and work properly with minimal deformities. Therefore, depending on several factors such as time and what condition of extreme exposure it is, denaturation can be both permanent and temporary in proteins.

2. 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. This 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.

Fig: Causes or Denaturation of proteins

Denaturing Agents:

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The most common factors that denature proteins includes:

1. Changes in temperature:

Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.

Example:

The proteins in eggs denature and coagulate during cooking. Other foods are cooked to denature the proteins to make it easier for enzymes to digest them.Medical supplies and instruments are sterilized by heating to denature proteins in bacteria and thus destroy the bacteria.

2. Changes in pH :

Most proteins at physiological pH are above their isoelectric points and have a net negative charge. When the pH is adjusted to the isoelectric point of the protein, its net charge will be zero. Charge repulsions of similar molecules will be at minimum and many proteins will precipitate. Even for proteins that remain in solution at their isoelectric points, this is usually the pH of minimum solubility.

If the pH is lowered far below the isoelectric point, the protein will lose its negative and contain only positive charges. The like charges will repel each other and prevent the protein from aggregating as readily. In areas of large charge density, the intramolecular repulsion may be great enough to cause unfolding of the protein. This will have an effect similar to that of mild heat treatment on the protein structure. In some cases the unfolding may be extensive enough to expose hydrophobic groups and cause irreversible aggregation. Until this occurs such unfolding will be largely reversible.

Some proteins contain acid labile groups and even relatively mild acid treatment may cause irreversible loss of function. This generally results from the breaking of specific covalent bonds and thus should be considered separately from denaturation. Exposure to strong enough acid at elevated temperatures will first release amide nitrogen from glutamine and asparagine groups and eventually lead to hydrolysis of peptide bonds.

The effects of high pH are analogous to those of low pH. The proteins obtain a large negative

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charge which can cause unfolding and even aggregation. The use of high pH to solubilize and alter protein structure is very important to the formation of fibers from proteins of plant origin

A number of reactions can cause chemical modification of proteins at alkaline pH's that are commonly encountered in protein processing. Many of these involve cysteine residues. Perhaps the most important are the base catalyzed beta eliminations of sulfur to yield dehydroalanine which can react with lysine to form lysinoalanine. This results in a loss of nutritive value of the protein and the products of the reaction may be toxic. Exposure of protein molecules to high pH should be minimized as much as is possible. Exposure to very high pH at elevated temperatures results in alkaline hydrolysis of peptide bonds.

3. Heavy Metal Salts:

Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt.

This reaction is used for its disinfectant properties in external applications. For example AgNO 3

is used to prevent gonorrhea infections in the eyes of new born infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds.

Mercury salts administered as Mercurochrome or Merthiolate have similar properties in preventing infections in wounds.

This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body.

Example:

Heavy Metal Salts Disrupt Disulfide Bonds:

Heavy metals may also disrupt disulfide bonds because of theirhigh affinity and attraction for sulfur and will also lead to thedenaturation of proteins.

4. Reducing Agents Disrupt Disulfide Bonds:

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Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. Review reaction. Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. Both of these examples are exhibited by the insulin in the graphic on the left.

If oxidizing agents cause the formation of a disulfide bond, then reducing agents, of course, act on any disulfide bonds to split it apart. Reducing agents add hydrogen atoms to make the thiol group, -SH. The reaction is:

Fig: Reducing agents Disrupts disulfide bond

5. Detergents:

Detergents are amphiphilic molecules (both hydrophobic and hydrophilic parts).

Example:

Disrupt hydrophobic interactions:

o hydrophobic parts of the detergent associate with the hydrophobic parts of the protein (coating with detergent molecules)

o hydrophilic ends of the detergent molecules interact favorably with water (nonpolar parts of the protein become coated with polar groups that allow their association with water)

o hydrophobic parts of the protein no longer need to associate with each other

o Dissociation of the non-polar R groups can lead to unfolding of the protein chain (same effect as in nonpolar solvents).

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6. Agitation:

Whipping action stretches the polypeptide chain until the bonds break.

Example:

Whipping of cream or egg whites

7. Alcohol Disrupts Hydrogen Bonding:

Hydrogen bonding occurs between amide groups in the secondary protein structure. Hydrogen bonding between "side chains" occurs in tertiary protein structure in a variety of amino acid combinations. All of these are disrupted by the addition of another alcohol.

A 70% alcohol solution is used as a disinfectant on the skin. This concentration of alcohol is able to penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell. A 95% alcohol solution merely coagulates the protein on the outside of the cell wall and prevents any alcohol from entering the cell. Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains.

In the prion protein, tyr 128 is hydrogen bonded to asp 178, which cause one part of the chain to be bonding with a part some distance away. After denaturation, the graphic show substantial structural changes.

Fig: Denaturation by alcohol

8. Denaturation by Acids and Bases:

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Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect.

As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk.

Example:

The denaturation reaction on the salt bridge by the addition of an acid results in a further straightening effect on the protein chain as shown in the graphic on the left.

Fig: Denaturation by acid and bases

How denaturation occurs at levels of protein structure:

Denaturing occurs when that shape is compromised and the molecule can no longer function in its desired capacity. Proteins may be denatured at the secondary, tertiary and quaternary structural levels, but not at the primary structural level.

1. In Primary Structure: the sequence of amino acids held together by covalent peptide bonds, is not disrupted by denaturation.

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2. In Secondary Structure : denaturation, proteins lose all regular repeating patterns such as alpha-helices and beta-pleated sheets, and adopt a random coil configuration

3. In Tertiary structure : denaturation involves the disruption of:

Covalent interactions between amino acid side-chains (such as disulfide bridges between cysteine groups)

Noncovalent dipole-dipole interactions between polar amino acid side-chains (and the surrounding solvent)

Van der Waals (induced dipole) interactions between nonpolar amino acid side-chains.

4. In quaternary structure : Denaturation, protein sub-units are dissociated and/or the spatial arrangement of protein subunits is disrupted.

Denaturations occurs at levels of protein structure

Measuring Protein Denaturation:

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Protein denaturation is commonly defined as any noncovalent change in the structure of a protein. This change may alter the secondary, tertiary or quaternary structure of the molecules. When using this definition it should be noted that what constitutes denaturation is largely dependent upon the method utilized to observe the protein molecule. Some methods can detect very slight changes in structure while other require rather large alterations in structure before changes are observed.

1. Loss of Solubility:

One of the oldest methods utilized to follow the course of denaturation was to measure changes in solubility. Changes in solubility might be evident in simple buffers or they might exhibit themselves only after exposure to other conditions, eg. 0.25M ammonium sulfate. Proteins vary greatly in their resistance to insolubilization by a variety of procedures and some proteins that are very important in foods are insoluble in their native state. The loss of solubility is only one of the last stages in a series of changes in structure that must have occurred. As such, this is a rather crude measure of protein denaturation.

In another sense however, the loss of solubility can be related to the loss of a great number of desirable characteristics of the protein. In many cases in food systems, most structural changes other than loss of solubility are unimportant and the role of many process designs and food additives is to maintain protein solubility.

When more sophisticated techniques are utilized many changes in protein structure that eventually result in a loss of solubility can be detected. In these cases the loss of solubility is more properly regarded as an effect of denaturation rather than as a measure of denaturation. To a consumer or a product development scientist who only observes that feathering occurs when some products are utilized to whiten coffee, loss of solubility, however, is the only event that matters. In the rest of this chapter, loss of solubility will be considered as an effect of denaturation.

2. Increased Proteolysis :

Most native proteins are quite resistant to the action of proteolytic enzymes. During digestion, proteins are exposed to extremes of pH to alter their structures in such a way as to expose the proper groups to enzyme molecules.

For some time, it has been known that a variety of procedures that alter protein's structures make them more susceptible to proteolysis. The rate and extent of proteolysis can be utilized as an indicator of protein denaturation.

In many cases, increases in proteolysis, like decreases in solubility, are the result of many changes in protein structure. In a series of experiments on ribonuclease, Burgess and Scheraga exposed this protein to a variety of combinations of pH and temperature. The molecule was then mixed with one of three different proteases. Under conditions of mild denaturation, they were able to observe which portions of

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the molecule were made susceptible to proteolysis first. Increasingly harsh treatments exposed other portions of the molecule to the action of the proteases. From these observations and knowledge of the tertiary structure of the molecule, they were able to hypothesize a pathway for the thermal denaturation of ribonuclease. This pathway was assumed to be the reverse of the pathway for protein folding, but there was no evidence for this to be the case.

3. Loss of Biological Activity

For those proteins that are enzymes, denaturation can be defined as the loss of enough structure to render the enzyme inactive. Changes in the rate of the reaction, the affinity for substrate, pH optimum, temperature optimum, specificity of reaction, etc., may be affected by denaturation of enzyme molecules.

Loss of enzymatic activity can be a very sensitive measure of denaturation as some assay procedures are capable of detecting very low levels of product. In some cases the loss of activity can be shown to occur only after some other changes in structure can be observed by other procedures. There may technically, then be denaturation of the protein before loss of activity occurs.

Enzymes are extremely important in the processing and preparing of food products. Processors may variously want to encourage or inhibit the activity of selected enzymes. In these cases, losses of activity may well be the only index of protein denaturation that is of interest.

A number of protein molecules may exhibit biological activities that are not enzymatic in nature. Antibodies for instance are capable of interacting with specific antigen molecules. Other proteins, like hemoglobin, may function as carriers while some, eg. ferritin, may function in the storage of specific components. The loss of any of these activities can be measured as protein denaturation.

4. Tritium-Hydrogen Exchange:

When compounds that contain tritium are placed in water they will rapidly exchange the tritium for normal hydrogen if the groups containing the tritium are exposed to the water. Tritium may be incorporated into proteins by a number of procedures. Probably the most common in exchange experiments involves the unfolding of the protein molecule in a medium where all of the water has been replaced by tritium oxide. When the protein is removed to a normal aqueous environment, three classes of tritium are often observed. Any tritium that is on the surface of the molecule along with any other that is not necessarily always on the surface, but that comes into contact with the surface under the conditions of study, will rapidly be lost from the molecule.

A second class of tritium molecules will be lost only when conditions that lead to partial protein unfolding occur. These are the class that can be utilized to monitor the rate and extent of denaturation. There may also exist a set of tritium molecules that are located in positions that are

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accessible to solvent only when the protein molecule is completely unfolded. The second group of tritium atoms do not exchange with the solvent because they are not exposed to the water. Such molecules must be located on the interior of the protein. If denaturation results in unfolding of the molecule and exposure of previously buried tritium groups to the solvent, exchange will occur. This procedure has been utilized quite extensively to study the mechanisms of stabilization of protein structure by small molecules.

5. Spectroscopic Procedures:

A variety of procedures have been developed that measure the interaction of electromagnetic radiation with molecules. Some of these procedures have proven to be very useful in the study of protein denaturation.

One such procedure is ultraviolet adsorption spectroscopy. This simply measures the wavelength of and the amount of ultraviolet radiation absorbed by a molecule. In proteins, both the wavelength and extent of absorption depend on the amino acids present and on their physical environments. There are a large number of such groups in a protein molecule and thus its U.V. spectrum quite often lacks detail. Under some circumstances however, these groups can absorb at a low wavelength, generally in the U.V., and then emit light at a larger wavelength. This process is known as fluorescence and is quite sensitive to the environment of the groups involved.

Both ultraviolet and fluorescence spectroscopy have been utilized to follow changes in the environments of various groups within protein molecules. Such changes in environment reflect changes in protein structure and thus denaturation.

The interaction of polarized light with protein can be measured by the techniques of circular dichroism and optical rotatory dispersion. These methods yield an indication of the extent of repeating structures present in protein and are generally utilized to give estimates of the amount of secondary structure present, eg. Alpha-helix, beat sheet or coil. While these procedures do not yield very precise estimates of the exact secondary structure of proteins, they are very useful for observing changes.

Denaturation at Interfaces:When proteins are exposed to either liquid-air or liquid-liquid interfaces, denaturation can occur.

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As a liquid-liquid interface, the protein comes into contact with a hydrophobic environment. If allowed to remain at this interface for a period of time proteins will tend to unfold and place as many of their hydrophobic groups as possible in the non-aqueous layer while maintaining as much charge as possible in the water layer.

To understand why protein unfolds at hydrophobic interfaces, it must be realized that the tertiary structure of a protein is not rigid. There are continued fluctuations about an average configuration. Any change in conformation that yields a higher energy state will spontaneously go back to the state of lowest energy. As a part of this process, hydrophobic groups will occasionally be positioned so that they have increased contact with the aqueous phase. When this occurs, these groups will assume the configuration of lowest free energy and will be removed from the water. If a hydrophobic group is exposed while a protein is in contact with a polar solvent, these groups will find a state of lower energy exists if they enter into the solvent phase. This will continue to occur until random fluctuations in protein structure can no longer yield a configuration of lower free energy.

The amount of unfolding that occurs at such an interface will depend on how rigid the three-dimensional protein structure is an on the number and location of hydrophobic groups in the molecule. A flexible, non-crosslinked protein will be able to unfold easier than will a highly structured and crosslinked one. If energy is applied to cause shear, the process will be accelerated. The shear can cause the protein to unfold, thus exposing its hydrophobic groups to the nonaqueous phase. It can also increase the interfacial area between the two phases and allow more proteins to come into contact with the nonaqueous phase.

This unfolding is essentially non-reversible because of the large energy barriers. Even if the phases should separate and the protein is forced into the aqueous phase the protein will not regain its original structure. Rather an association of hydrophobic groups will cause the protein to aggregate.

The same forces are in operation when a protein migrates to a liquid-air interface. Hydrophobic groups tend to associate in the air and the protein unfolds. The presence of shear causes to help unfold the protein and to introduce more air into the solution. Both of these effects can be minimized by keeping the temperature low (to weaken hydrophobic bonds) and by minimizing the interfacial area. If the interface is limited, then only a small amount of protein will be able to denature. The presence of this denatured protein will serve as a barrier to further denaturation. Proteins are often utilized in food products to stabilize emulsions or to incorporate air. These cases will be examined in more detail when emulsions and foams are discussed.

Benefits of Denatured Proteins:

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When bodybuilding or simply working out, denatured protein--and any form of protein, really--is nutritionally beneficial. After you finish a workout, your body has to absorb new protein in order to repair and add any mass to the muscles that you just used. Whether you take a whey protein, a denatured protein or another form of protein, protein digestion takes place in the stomach. Because denatured protein is already broken apart structurally, it will be able to get to your muscles quickly. The protein that you put into your body will be used to maximize your workout by rebuilding your muscles and giving them the energy they need to come back stronger.

Example: Denaturing of milk proteins:

1.Casein:

It undergoes little if any denaturing by heat or acid because it doesn’t have the usual secondary

and tertiary protein structures. In the stomach, under the action of the stomach acids, casein

forms glue-like cloths, which are difficult to digest. For that reason it stays in the stomach

longer, which gives it the slow-release properties that are popular among bodybuilders.

We now know that casein releases certain bio-active peptides under the digestive powers of the proteolitic (protein-digesting) enzymes in the stomach, like pepsin, trypsin and chymotrypsin. These enzymes break down the longer protein fractions to 2 – 20 amino acids-long peptides that exhibit multiple health benefits.

2. Whey:

Whey is also very popular among those who value its properties to release amino acids in the

blood stream in a very short period of time, making it an ideal protein for recovery, muscle

building and re-building.

Whey protein also exhibits health benefits due to its native bioactive fractions, among them beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin and lactoferrin. All these bioactive fractions are shown to have cardiovascular, digestive, endocrine, immune and nervous system- modulating effects But, only when they are in their undenatured form.

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