CHEMISTRY OF MILK

Module 3. Milk proteins

Lesson 11
PROTEIN DENATURATION AND HYDROLYSIS

11.1 Introduction

Proteins in milk play an important role in the human nutrition. The processing and handling of the milk will change the physico chemical environment which would result in the denaturation of the protein molecule. As such knowledge of these changes is necessary for having a comprehensive idea about the behaviour of the proteins in the changed environment.

11.2 Definition

Denaturation is a phenomenon that involves transformation of a well-defined, folded structure of a protein, formed under physiological conditions, to an unfolded state under non-physiological conditions.

11.3 Agents Causing Protein Denaturation

Change in the structure of proteins can be caused by a variety of factors. Some of these are encountered frequently while others are more of theoretical interests. A change in the structure of protein by heat, acid, alkali, or other agents such as sound waves, surface forces, pressure, UV radiation and ionizing radiations, results in loss of solubility and coagulation which is otherwise known as denaturation. Treatment with organic solvents such as alcohol, acetone, and solutes like urea, guanidine and ionic detergents would also result in protein denaturation. It is normally irreversible. Denatured proteins lose their biological activity (e.g. as enzymes), but nott heir nutritional value. Indeed, their digestibility is improved compared with the native structures, which are relatively resistant to enzymatic hydrolysis.

11.4 Thermal Denaturation

When proteins are exposed to increasing temperature, losses of solubility or enzymatic activity occurs over a fairly narrow range. Depending upon the protein studied and the severity of the heating, these changes may or may not be reversible. As the temperature is increased, a number of bonds in the protein molecule are weakened. The first affected are the long range interactions that are necessary for the presence of tertiary structure. As these bonds are first weakened and are broken, the protein obtains a more flexible structure and the groups are exposed to solvent. If heating ceases at this stage the protein should be able to readily refold to the native structure. As heating continues, some of the cooperative hydrogen bonds that stabilize helical structure will begin to break. As these bonds are broken, water can interact with and form new hydrogen bonds with the amide nitrogen and carbonyl oxygen of the peptide bonds. The presence of water further weakens nearby hydrogen bonds by causing an increase in the effective dielectric constant near them. As the helical structure is broken, hydrophobic groups are exposed to the solvent.

The effect of exposure of new hydrogen bonding groups and of hydrophobic groups is to increase the amount of water bound by the protein molecules. The unfolding that occurs increase the hydrodynamic radius of the molecule causing the viscosity of the solution to increase. The net result will be an attempt by the protein to minimize its free energy by burying as many hydrophobic groups while exposing as many polar groups as possible to the solvent. While this is analogous to what occurred when the protein folded originally, it is happening at a much higher temperature. This greatly weakens the short range interaction that initially direct protein folding and the structures that occur will often be vastly different from the native protein. Upon cooling, the structures obtained by the aggregated proteins may not be those of lowest possible free energy, but kinetic barriers will prevent them from returning to the native format. Any attempt to obtain the native structure would first require that the hydrophobic bonds that caused the aggregation be broken. This would be energetically unfavorable and highly unlikely. Only when all the intermolecular hydrophobic bonds were broken, could the protein begin to refold as directed by the energy of short range interactions.

The exposure of this large number of hydrophobic groups to the solvent, however, presents a large energy barrier that make such a refolding kinetically unlikely. Exposure of most proteins to high temperatures results in irreversible denaturation. Some proteins, like caseins, however, contain little if any secondary structure and have managed to remove their hydrophobic groups from contact with the solvent without the need for extensive structure. This lack of secondary structure causes these proteins to be extremely resistant to thermal denaturation.

The increased water binding noted in the early stages of denaturation may be retained following hydrophobic aggregations. The loss of solubility that occurs will greatly reduce the viscosity to a level below that of the native proteins.

11.5 Effect of pH on Protein Denaturation

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 readily aggregating. 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 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 result in a loss of nutritive value of the protein and the products of there action 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.

11.6 Changes Dielectric Constant

The addition of a solvent that is miscible with water, but that is less polar will lower the dielectric constant of the system. This will tend to increase the strength of all electrostatic interactions between molecules that were in contact with water. Many of the protein hydrogen bonds are effectively removed from the solvent and will not be affected. The presence of the less polar solvent will also have the effect of weakening the hydrophobic bonds of the proteins. These bonds depend upon an increase in the order of water when they are broken for their existence. As there is less water in the system, this becomes less important and at some level of replacement, these groups are at a lower energy level when in contact with the solvent. The structure of the protein will be changed and hence, it will be denatured. The reversibility of the process depends to a large extent on the nature of the non-polar solvent, the extent of unfolding the temperature of the system and the rate of solvent removal. When large amounts of the solvent are present, the protein will be largely unfolded with extensive exposure of the hydrophobic groups. If the protein could be instantaneously transferred to pure water at room temperature, the protein would most likely aggregate and precipitate. The sudden exposure of the hydrophobic groups to water would cause them to try to remove themselves from the aqueous phase as soon as possible. Even before the short range interactions could redirect the folding of the protein aggregation would occur. If the solvent exchange were slow, there would be a better chance that the hydrophobic groups would be able to return to the interior of the molecule and prevent aggregation. If the exchange occurred at low temperatures, the chances of regaining the native structure would be even better. At low temperatures, the hydrophobic groups may in part be stable in the aqueous phase or at least not as unstable. In this case, the removal of the solvent has little affect. When the temperature is subsequently increased, the normal course of protein refolding can occur. Solvent precipitation is often utilized as a means of purifying and concentrating enzymes. It is extremely important that both the solvent and the protein solution be cold when they are mixed and that the subsequent removal of the solvent be performed at reduced temperature. This helps to insure the recovery of enzyme activity.

11.7 Denaturation at Interfaces

When proteins are exposed to either liquid-air or liquid-liquid interfaces, denaturation can occur. 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 on the number and location of hydrophobic groups in the molecule. A flexible, non-cross linked protein will be able to unfold easier than will a highly structured and cross linked 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 non - aqueous phase. It can also increase the interfacial area between the two phases and allow more proteins to come into contact with the non-aqueous 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 helps to 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.

11.8 Ionic Strength

Proteins are usually more soluble in dilute salt solutions than in pure water. The salts are thought to associate with oppositely charge groups in the protein. This combination of charged groups binds more water than do the charged groups alone and protein hydration is increased. With most proteins there is little change in solubility as more salt is added until some very high salt content is reached. At very high levels of salt there is a competition between the ions and the proteins for water of hydration. When the salt concentration is high enough, the proteins will be sufficiently dehydrated to lose solubility. Removal of the salt or dilution to a low enough concentration will usually result in the recovery of native structure.

11.9 The Effect of Protein Cross Linkers

The presence of groups that cross link protein molecules will tend to lower the extent of protein denaturation. There are two main reasons for this type of behavior. First, when proteins are cross linked it is more difficult for them to unfold. As energy is added to the system and secondary bonds are weakened, the presence of cross linkers will tend to maintain structure. This is especially true if the cross links are covalent as in the case of disulfide bonds. The more compact the molecule is and the greater the number of disulfide linkages present, the greater the stability of the protein. While secondary forces may be weakened and some bonds can be broken, the cross linkers will tend to keep these groups in fairly close proximity. They also tend to prevent the exposure of large numbers of hydrophobic groups to the solvent. When conditions are returned to the native state, there is now a much greater chance for the proper secondary interaction to occur and for the protein to assume the native configuration.

A second effect has to do with the differences in entropy between the native and unfolded states. If a protein can be caused to assume a completely random coil conformation, there will be a large increase in entropy compared to the native structure. This entropy must be overcome if the protein is to refold into a native conformation. When cross linking groups are present, a completely random coil conformation can not be assumed. These groups introduce order into the structure and there is a considerable loss in the amount of disorder that can be achieved in the most denatured state. Because of this, the entropy change between the native and denatured state is not nearly as great and there will be less of a driving force for denaturation. If the cross linking groups are broken before denaturation and thus allowed to randomly form after denaturation, no stability will be added to the protein by the pressure of these groups.

No reactions involving primary covalent bonds (such as peptide linkages) occur during denaturation the unfolding of the molecules often exposes groups which may undergo chemical reactions (oxidation of sulfhydryl groups by the atmospheric oxygen).

11.10 Hydrolysis of Protein

Proteins are polymers of amino acids which are held by the polypeptide bonds between them. When these bonds are broken the protein splits in to smaller peptide and may even proceed to that extent of release for amino acids or smaller peptides. For these reactions, the presence of a suitable agent, or enzymes. The solution containing these smaller peptides and even amino acids is called hydrolysate solution. This process will help the digestion of the proteins and also study of the various amino acids present in the protein.