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Introduction Protein is one of the four major constituents of food. Some of the ways that proteins contribute to foods are listed below

Nutritional

• supply amino acids which are required for protein synthesis

• source of carbon, nitrogen, sulfur which are used in metabolism

Functional

• contribute to food systems through their ability to form foams, gels, or by stabilization of emulsions

Catalytic

• enzymes are responsible for nearly all of the biochemical processes that occur in biological systems (the rest are catalyzed by ribozymes)

Structural

• fibrous structures in meat, cheese curd, and the texture of bread are due to a large extent to protein interactions

Sensory

• colour and flavour from Maillard reaction products that arise from free amino and reducing groups

• peptides and free amino acids contribute flavour to food

It is necessary to understand the nature of the basic building blocks of proteins - amino acids - and the way these interact to form three-dimensional proteins, before we can begin to understand how proteins function in foods. Being able to predict protein structure-function relationships, to enhance functional properties, formulate new products, or improve existing products are some of the goals of protein chemists.

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Amino Acids

Basic Structural Unit of Proteins Amino acids are the basic building blocks of proteins. An amino acid consists of an amino group, a carboxyl group, a hydrogen atom and a unique R-group attached to the center carbon.

Amino acids are "bricks" constitutive of proteins.

An amino acid carries simultaneously :

a carboxylic acid function -COOH, which is a weak acid (2< pKa< 2.5)

an amine function -NH2, which is a weak base (9< pKa< 9.5).

In solution as well as in the solid state, the proton of the carboxylic acid group is transferred onto the amine to give a neutral entity, called zwitterion.

Under these form, amino acids can be considered as being salts of the weak acid -COO- and the weak base -NH3

+ and therefore they behave as amphoteric particles.

Les acides aminés, sous forme de sel de l'acide faible -COO- et la base faible -NH3+ ont donc un

comportement amphotère.

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20 Common Amino Acids There are only 20 common amino acids that are used to make up proteins, but keep in mind that there are hundreds of different amino acids found in nature. Amino acids can be categorized according to the chemical nature of the R-group.

Amino acids with non-polar R-groups

• [Aliphatic Group] • [Aromatic R-Group] • [Sulfur containing R-Group]

Amino acids with non-polar R-groups Aliphatic Group

Amino Acid

Abbreviation Structure Points of Note

Glycine Gly (G)

Alanine Ala (A)

Valine Val (V)

Glycine is the only amino acid which has a prochiral carbon, since the R-group is a proton. Glycine does not exist in D & L forms like the other amino acids.

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Leucine Leu (L)

Isoleucine Ile (I)

Proline Pro (P)

Proline differs from other common amino acids by having a cyclic R-group which is a secondary amino acid. It is referred to as an 'imino' acid.

Aromatic R-Group

Amino Acid Abbreviation Structure Points of Note

Phenylalanine Phe (F)

These amino acids are predominantly non-polar in nature although the hydroxyl group of tyrosine makes it less hydrophobic. It is the absorption of ultraviolet light by the aromatic ring that allows proteins containing these amino acids to be detected at 280 nm.

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Tyrosine Tyr (Y)

Tryptophan Trp (W)

Sulfur Containing R-Group


Cysteine Cys (C)

Two cysteine residues can form a covalent disulfide bond, and contribute to the structure of proteins. The release of sulfur containing compounds, originating from these amino acids can contribute to desirable or undesirable odours in foods.

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Methionine Met (M)

Amino acids with polar R-groups

• [Hydroxyl R-Group] • [Acidic R-Group] • [Amide R-Group] • [Basic R-Group]

Amino acids with polar R-groups Hydroxyl R-Group


Serine Ser (S)

These two amino acids are potential sites for the enzymatic addition of sugars to proteins, referred to as O-glycosylation.

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Threonine Thr (T)

Acidic R-Group


Aspartate Asp (D)

Glutamate Glu (E)

These R-groups are almost always negatively charged under physiological conditions.

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Amide R-Group


Asparagine Asn (N)

Glutamine Gln (Q)

The amide group of asparagine is a potential site for the enzymatic addition of sugars to proteins, referred to as N-glycosylation.

Glutamine residues may be acted on by an enzyme called transglutaminase, which catalyzes the cross-linking between glutamine and a primary amino group.

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Basic R-Group


Lysine Lys (K)

Arginine Arg (R)

The side chain of lysine and arginine are positively charged under physiological conditions. Histidine, however, can be charged or uncharged, and may act as a buffer at neutral pH. Lysine is important in food systems since the free amino group can participate in Maillard browning reactions, and contribute to the colour and flavour of cooked foods.

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Histidine His (H)

Taken from: http://www.agsci.ubc.ca/courses/fnh/410/modules.htm

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Separation of amino acids by electrophoresis Each amino acid has a specific isoelectric pH (pHi the pH at which amino acids are electrically neutral). At pH differing from pHi, amino acids bear an overall charge and therefore migrate under the effect of an electric field. Thus, working at a fixed pH (buffered solution) allows one to separate various amino acids by electrophoresis.

When an amino acid is placed in an electric field, it migrates towards the electrode of opposed polarity, while the neutral (zwitterionic) molecules do not migrate.

Therefore :

When pH > pHi the amino acid bears a negative total charge: it migrates towards the positive electrode (anode).

When pH < pHi the amino acid bears a positive total charge: it migrates towards the negative electrode (cathode).

When pH = pHi the amino acid is in its zwitterionic, neutral form: it does not migrate and remains at the starting point.

The pH of the solution is adjusted in such a way that an optimal separation occurs.

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Titration Curve & pKa Of the twenty common amino acids, thirteen have two ionizable groups, the alpha-carboxyl and the alpha-amino group. The remaining seven amino acids have three ionizable groups, since their R-group can also be ionized. The pH of the environment of the amino acid and the pKa of the ionizable groups will determine the charge associated with an amino acid. The pKa value of an ionizable group is the pH at which there are equal amounts of protonated and unprotonated species. At a pH below the pKa value, a group will be mainly protonated, while at a pH above the pKa, a group will be mainly unprotonated. The carboxyl group and the amino group have very different pKa values.

pK Values of Ionizable Groups in Protein

Group Typical pK*

Terminal carboxyl 3.1

Aspartic and

glutamic acid

4.4

Histidine

6.5

Terminal amino

8.0

Cysteine 8.5

Tyrosine

10.0

Lysine

10.0

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Arginine

12.0

* pK values depend on temperature, ionic strength, and the microenvironment of the ionizable group.

A pKa value for an ionizable group can be determined by monitoring the pH of a solution as equivalents of a base such as NaOH are added. Such a graph is called a titration curve. The pH values on the titration curve where an inflection point occurs are the pKa values. The titration curve shows how the charge varies with pH.

If you wish to further explore the area of amino acid titration, you can visit the following web site: http://cti.itc.Virginia.edu/~cmg/Demo/script_frame.html Taken from: [http://chimge.unil.ch/En/ph/1ph81.htm]

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Levels of Protein Structure

Primary Structure

Four levels of structure are frequently sited in discussion of protein architecture. Primary structure is the amino acid sequence and the location of disulfides, if there are any. The primary structure is thus a complete description of the covalent connections of a protein.

Secondary Structure

Secondary structure refers to the special arrangement of amino acid residues that are near one another in the linear sequence. Some of these steric relationships are of regular kind, giving rise to a periodic structure. Proteins are not randon long chains but rather are defined compact structures.

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The alpha-helix and beta-pleated sheet and collagen helix are elements of secondary structure. Limits on this type of structure ranges from steric hinderance of bulk R-groups to the presence of an imino acid.

Quiz question:

What is the name of the imino acid commonly found in proteins? Proline.

(If you didn't know this please review the information on amino acids)

Ribbon Model: Alpha Helix is shown in Brown, B-sheets in purple,

disulfide bridges in yellow.

Tertiary Structure

Tertiary structure refers to the spatial arrangement of amino acid residues that are far apart in the linear sequence. It is the folding of the amino acid structure on to itself. Usually there are hydrophilic residues on the surface and hydrophobic residues in the interior of the protein.

Quaternary Structure

Quaternary structure refers to the spatial arrangement of tertiary structures and the nature of their contacts. The constituent chains of a multisubunit protein can be identical or different. In basic terms, quaternary protein structures are made up of more than one tertiary unit. Quaternary structure can be regulatory or catalytic chains and can be synthesized as separate gene products or processed (proteolyzed) after the synthesis of a single chain.

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If you wish to further explore the area of protein structure, you can visit the following web sites:

http://cti.itc.Virginia.edu/~cmg/Demo/script_frame.html http://monera.ncl.ac.uk/protein/protein.html http://ull.chemistry.uakron.edu/genobc/Chapter_19/

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Bonds Involved in Protein Structure

Hydrogen Bonds

Hydrogen bonding occurs between a partial negative charge and the partial positive charge of molecules containg the hydrogen atom. In the alpha-helix, each hydrogen is formed between the hydrogen atom attached to the electronegative nitrogen atom of one peptide linkage and the oxygen linkage of the carbonyl of the fourth amino acid behind it.

Hydrophobic Bonds The sight of dispersed oil droplets coming together in water to form a single large oil drop is a familiar one. An analogous process occurs at the atomic level: nonpolar molecules or groups tend to cluster together in water. These associations are called hydrophobic attractions. In a figurative sense, water tends to squeeze nonpolar molecules together.

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Disulfide Bonds

Cross-links between chains or parts of chain are called disulfide bonds formed by the oxidation of the cysteine residues. The resulting disulfide is called cystine. Usually, extracellular proteins contain several disulfide bonds whereas intracellular proteins often lack them.

Ionic bonds

Electrostatic bonds consist of a charged group which can attract an oppositely charged group. This kind of attraction is also known as an ionic bond, salt linkage, salt bridge, or ion pair. Like ionic charges can also be repulsive.

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Van der Waals Forces

Van der Waals bonds are nonspecific attractive forces, also referred to as temporary dipole interactions, and are weaker than hydrogen and electrostatic bonds.

A single Van der Waal bonds counts for very little because the attraction is weak. Furthermore, these forces fade rapidly when a distance between a pair of atoms becomes even 1 Angstrom greater than their contact distance. It becomes significant only when numerous atoms are involved.

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Denaturation of Proteins - Physical Influences

Protein Denaturation: Temperature

Denaturation can be defined as any modification of secondary, tertiary, or quaternary structure of the protein molecule, excluding breakage of covalent bonds. Denaturation is therefore a process by which hydrogen bonds, hydrophobic interactions and salt linkages are broken and the protein is unfolded.

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 at the transition midpoint, where the concentration ratio of native and denatured states is 1, is known either as the melting temperature (Tm), or the denaturation temperature (Td).

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[Fig. 1 Schematic illustration of the denaturation of a protein molecule] (Source: Fennema, OR. 1976. Principles of Food Science. Marcel Dekker, Inc., New York. chapter 5.)

Mechanism

The mechanism of temperature-induced denaturation is highly complex and involves primarily destabilization of the major noncovalent interactions. Hydrogen bonding, electrostatic, and van der Waals interactions are exothermic in nature. Therefore, they are destabilized at high temperatures and stabilized at low temperatures.

The theoretical curve for the amount of a protein denatured at a given temperature in 10 min is shown in Figure 2. Note that at only 5oC above the 50% denaturation temperature, barely 1 % remains, but at 5oC below, as much as 8% is lost.

[Fig. 2 Theoretical diagram showing the percent of protein remaining undenatured after 10 min incubation.] (Source: Scopes, RK. 1994. Protein Purification: Principles and Practice. 3rd ed. Springer-Verlag New York, Inc. USA. P. 95-101.)

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Factors

Susceptibility of proteins to heat denaturation depends on a number of factors. Water greatly facilitates thermal denaturation of proteins. Dry protein powders are extremely stable to thermal denaturation. The value of Td decreases rapidly as the water content is increased from 0 to 0.35 g water/g protein. An increase in water content from 0.35 to 0.75g water/g protein causes only a marginal decrease in Td. Above 0.75g water/g protein, the Td of the protein is the same as in dilute protein solution. Some proteins are stable at temperatures as high as 100oC if the moisture content is very low.

Additive such as salts and sugars affect thermostability of proteins in aqueous solutions. Sugars such as sucrose, lactose, glucose, and glycerol stabilize proteins against thermal denaturation. Addition of 0.5 M NaCl to proteins such as b-lactoglobulin, soy proteins, serum albumin, and oat globin significantly increases their Td.

The amino acid compostition also affects thermal stability of proteins. Proteins that contain a greater proportion of hydrophobic amino acid residues, especially Val, Ile, Leu, and Phe, tend to be more stable than the more hydrophilic proteins. Other factors, such as disulfide bonds and the presence of salt bridges buried in hydrophobic clefts, may also contribute to thermostability.

Valine Isoleucine

Leucine Phenylalanine

In addition, ionic strength, pH, and type of ions present in solution also affect the susceptibility of proteins to heat denaturation.

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MEAT

During cooking of flesh foods (meat, poultry, and fish), the final internal temperature of the product seldom exceeds a range of about 66-74oC. During the early stages of cooking (30-50oC), there is an unfolding of peptide chains, formation of relatively unstable crosslinkages, and partial denaturation of the sarcoplasmic proteins. These changes cause a tightening of the myofibrillar structure, resulting in toughness and decreased water-holding capacity. In the latter stages of pasteurization (50-74oC), new stable crosslinkages are formed in conjunction with denaturation and coagulation of both sarcoplasmic and myofibrillar proteins. At appropriate temperatures, collagen shrinks (61-63oC in meat and 45oC in fish) and softens as the ordered helical structure collapses. The overall result is tenderization of connective tissue and toughening of myofibrillar proteins.

Sources:

Fennema, OR. 1996. Food Chemistry. 3rd Edition. Marcel Dekker, Inc., New York. Chapter 6.

Scopes, RK. 1994. Protein Purification: Principles and Practice. 3rd ed. Springer-Verlag New York, Inc. USA. P. 95-101.)\

Protein Denaturation: Mechanical Treatment

High mechanical shear generated by shaking, whipping, etc. can cause denaturation of proteins. Many proteins denature and precipitate when they are vigorously agitated because of incorporation of air bubbles and adsorption of protein molecules to the air-liquid interface. Since the energy of the air-liquid interface is greater than that of the bulk phase, proteins undergo conformational changes 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. The nonpolar residues of denatured protein orient toward the gas phase and the polar residues orient toward the aqueous phase.

Several food processing operations involve high pressure, shear, and high temperature, for example, extrusion, high speed blending, and homogenization. When a high shear rate is produced by a rotating blade, subsonic pulses are created and cavitation also occurs at the trailing edges of the blade. Both of these events contribute to protein denaturation. The greater the shear rate, the greater is the degree of denaturation. The combination of high temperature and high shear force causes irreversible denaturation of proteins.

Source: Fennema, OR. 1996. Food Chemistry. 3rd Edition. Marcel Dekker, Inc., New York. Chapter 6.

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Protein Denaturation: Pressure

One of the thermodynamic variables that affect conformation of proteins is hydrostatic pressure. Unlike temperature-induced denaturation, which usually occurs in the range of 40-80oC at 1 atmospheric pressure (atm); pressure induced denaturation can occur at 25oC if the pressure is sufficiently great. Most proteins undergo pressure-induced denaturation in the range of 1-12 Kbar.

Pressure-induced denaturation of proteins occurs mainly because proteins are flexible and compressible. Although amino acid residues are densely packed in the interior of globular proteins, some void spaces exist and this leads to compressibility. The average partial specific volume of globular proteins in the hydrated state is about 0.74 ml/g. The larger the hydrated state of a protein, the larger is the contribution of void spaces to partial specific volume, and the more unstable the protein will be when pressurized. Fibrous proteins are mostly devoid of void spaces, and hence they are more stable to hydrostatic pressure than globular proteins.

Pressure-induced protein denaturation is highly reversible. Most enzymes, in dilute solutions, regain their activity once the pressure is decreased to atmospheric pressure. However, regeneration of near complete activity takes several hours.

High pressure processing applications

The application of high pressure to processing of foodstuffs is currently a subject of major interest for both food preservation (microbial inactivation) and food preparation.

Effects of pressure on cheese yield

Heat treatment has been used to promote whey protein denaturation and, consequently, increase its recovery in the curd. However, heating impaired the gel-forming properties of milk, led to longer coagulation times and weaker gels, and resulted in a practical limit of a 50% maximum of whey protein that could be incorporated. Unlike heat treatment, pressurization of milk to <400 Mpa improved milk coagulation properties. The denaturation and resulting incorporation of whey proteins into the curd effectively result in an increased in cheese yield, primarily by retention of moisture. Pressurization of milk for 30 min at 300 and 400 Mpa caused estimated mean curd weight increases of 20%, and a substantial 15% decrease in protein loss in the whey fraction. Although pressurization of milk for 30 min at 200 Mpa brought about a 20% denaturation of b-lactoglobulin, the curd weight only increased significantly above this pressure.

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Effects of pressure on cured meats

For meat products, high pressure treatment has been explored mainly in relation to the control of microorganisms, to the enzymatic tenderisation and to gel-setting. While the colour of minced beef has been found to be affected by pressure treatment as a result of globin denaturation and oxidation of myoglobin to metmyoglobin, nitrite was found to prevent such pressure-induced discoloration of minced meat. Cured meats may thus be the type of products which can benefit from high pressure technology.

Source: Fandino, RL. Carrascosa, AV and Olano, A. 1996. The effects of high pressure on whey protein denaturation and cheese-making properties of raw milk. J. Dairy Sci. 79:929-936.

Fennema, OR. 1996. Food Chemistry. 3rd Edition. Marcel Dekker, Inc., New York. Chapter 6.

Jensen, LB. and Skibsted, LH. 1996. High-pressure effects on oxidation of nitrosylmyoglobin. Meat Sci. 44(3):145-149.

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Protein Denaturation: Interfaces

Commonly, the boundary between a liquid and a gas is designated as a “surface,” whereas with other combinations of phases, the junction is termed an “interface.” Layering of proteins at an interface surface can result in denaturation. This results from unfolding of protein molecules along the interface, with hydrophilic residues remaining in the aqueous phase and hydrophobic residues tending to associate with the nonaqueous phase. Thus it is important, when attempting to preserve the native properties of proteins, to avoid creating interfaces, such as those in foam.

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[Fig. 3. Attractive forces between molecules at the surface, at the interface, and in the interior of the liquid phases]. Source: Fennema, OR. 1976. Principles of Food Science. Marcel Dekker, Inc., New York. chapter 5.

Denaturation of Proteins - Chemical Influences

Protein Hydration The contribution of water to protein structure

Hydration is very important for the three-dimensional structure and activity of proteins [472]. Indeed, they lack activity in the absence of water. In solution they possess a conformational flexibility, which encompasses a wide range of hydration states, not seen in the crystala or in non-aqueous environments. Equilibrium between these states will depend on the activity of the water within its microenvironment; i.e. the freedom that the water has to hydrate the protein [434]. Thus, protein conformations demanding greater hydration are favored by more (re-)active water (e.g. high density water containing many weak bent and/or broken hydrogen bonds) and 'drier' conformations are relatively favored by lower activity water (e.g. low-density water

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containing many strong intra-molecular aqueous hydrogen bonds).The folding of proteins depends on the same factors as control the junction zone formation in some polysaccharides; i.e. the incompatibility between the low-density water (LDW) and the hydrophobic surface that drives such groups to form the hydrophobic core.b Non-ionic kosmotropes, therefore, stabilize the structure of proteins. In addition, water acts as a lubricant, so easing the necessary peptide amide-carbonyl hydrogen bonding changes. Intramolecular peptide (amide) hydrogen bonding makes a major contribution to protein structure and stability but is only effective in the absence of accessible competing water. Even just the presence of close-by water molecules causes peptide hydrogen bonds to lengthen [524], so loosening the structure. Water molecules can bridge between the carbonyl oxygen atoms and amide protons of different peptide links to catalyze the formation, and its reversal, of peptide hydrogen bonding as well as forming long-lived linkages stabilizing protein-ligand and protein-protein interfaces (e.g. [688]). The internal molecular motions in proteins, necessary for biological activity, are very dependent on the degree of plasticizing, which is determined by the level of hydration. Thus internal water enables the folding of proteins and is only expelled from the hydrophobic central core when finally squeezed out by cooperative protein chain interactions [352]. The position of the ES CS equilibrium around enzymes has been shown to be important for their activity with the enzyme balanced between flexibility (CS environment) and rigidity (ES environment). Addition of non-ionic chaotropes only or kosmotropes only both inhibit the activity of enzymes (by shifting the equilibrium to the right or left respectively) whereas an intermediate mixture of kosmotropes and chaotropes restores optimum activity [276].

The first hydration shell around proteins (~0.3 g/g) is ordered; with high proton transfer rates and well resolved time-averaged hydration sites; surface water showing coherent hydrogen-bond patterns with large net dipoles [702]. As hydration sites may be positioned close together and therefore mutually exclusive, it has been argued that the solvation is better described as a water distribution density function rather than by specific water occupied sites. For example, no more than 164 of the 294 high-density hydration sites around myoglobin are occupied at any moment and there is no correlation between the maximum site density, occupancy and residence time [542]. The first hydration shell is also 10-20% denser than the bulk water and probably responsible for keeping the molecules sufficiently separated so that they remain in solution [315] (i.e. solutions are kinetically stable but often thermodynamically unstable). Although a significant amount of this density increase has been shown to be due to simple statistical factors dependent upon the way that the surface is defined in depressions [401], much is due to a protein's structure with the excess of polar hydration sites (tending to increase surface density) easily counteracting the remaining non-polar surface groups (tending to produce low density surface water). This water is required for the protein to show its biological function as, without it, the necessary fast conformational fluctuations cannot occur. Using X-ray analysis, the hydration shell shows a wide range of non-random hydrogen-bonding environments and energies. Proteins are formed from a mixture of polar and non-polar groups. Water is most well ordered round the polar groups where residence times are longer than around non-polar groups. Both types of group create order in the water molecules surrounding them but their ability to do this and the types of ordering produced are very different. Polar groups are most capable of creating ordered hydration through hydrogen bonding and ionic interactions (a excellent guide to amino acid hydrogen bonding is given elsewhere). This is most energetically favorable where there is no pre-existing order in the water that requires destruction. The water is slow to exchange, showing the dynamic behavior of bulk water 25°C colder [147]. Low-density water (such as ES) is promoted [148, 276] surrounding this dense hydration and polyelectrolyte double

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layer (as described in the 'Polysaccharide hydration' section). Non-polar groups promote clathrate structures [153] (such as ES) surrounded by denser water. It is no surprise, therefore that the degree of hydrophobic hydration is correlated with the hydration of the polar groups. Clathrate shells contain loosely held water with greater rotational freedom than in the bulk [139]. However, under favorable conditions clathrate hydrophobic hydration may exert pressure on non-polar C-H bonds pushing them in, so contracting their bond length and increasing their vibrational frequency. This 'push-ball' hydration [149] should not be thought of as 'typical' hydrogen bonding even if the CH···OH2 distances are suitably close. They can be considered as part of a continuum of hydrogen bonding behavior, however, where the OH2 behaves as a much more weakly interacting base than usual and the C-H behaves with reversed dipolar behavior compared with the more usual O-H hydrogen-bonding partners [625].

There are significant differences in the directional rates of water diffusion perpendicular and parallel to the protein surface that are maximal at about 6 Å but still determinable at 15 Å from the surface [542]. It is clear that evolutionary processes have made use of the organization in this water surrounding proteins to create preferred diffusive routes and orientation for metabolites and favored conformational changes and interactions. Such diffusive paths lead to binding sites with their own helpful hydration. It has been suggested that pressure waves formed from flickering water clusters (e.g. as described elsewhere) may link protein molecular vibrations, so carrying information through the intracellular milieu [549] and powering product movement between enzymes in biochemical pathways [665].

The energetic optimization of mutual hydrogen bonded networks between protein, water and ligand is an intrinsic part of the molecular recognition process in enzymes, binding proteins and biological macromolecules generally [412].

Note that water bound and oriented in empty ligand binding sites will reduce the entropy of activation when replaced by the ligand [414].

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Figure 1. The water network links secondary structures within the protein and so determines not only the fine detail of the protein's structure but also how particular molecular vibrations may be preferred. The above chain of ten water molecules, linking the end of one α-helix (helix 9, 211-227) to the middle of another (helix 11, 272-285) is found from the X-ray diffraction data of glucoamylase-471, a natural proteolytic fragment of Aspergillus awamori glucoamylase [155]. Figure 2. The above centrally-placed water molecule makes strong hydrogen bonds to residues in three separated parts of the ribonuclease molecule holding them together. This water molecule and its binding site are conserved across the entire family of microbial ribonucleases [345].

Water molecules have also proved integral to the structure and biological function of a dimeric hemoglobin [377]. The internal water molecules in proteins have been surveyed [725].

Protein folding is driven by hydrophobic interactions, due to the unfavorable entropy decrease (mostly translational [686]) forming a large surface area of non-polar groups with water. Consider a water molecule next to a surface to which it cannot hydrogen bond. The incompatibility of this surface with the low-density water that forms over such a surface [29] encourages the surface minimization that drives the proteins' tertiary structure formation. Such hydrophobic collapse is necessarily accompanied and guided by (secondary) structural hydrogen-bond formation between favorable peptide linkages in parallel with their desolvation [467]. A driving force for this, in crowded intracellular environments, is the release of water to be available for the hydration of other solutes. The folding route is controlled by the desolvation barriers [676] and aided and directed by water-mediated contacts. Similar factors help organize proteins involved in quaternary and equilibrium cluster formation, where each water-mediated interaction has been estimated to contribute an average of 4.4 kJ mol-1 to protein-protein interface stabilization [688]. Water is thus intimately involved in guiding protein folding and needs to be involved in protein structural prediction studies [643]. The importance of subtle hydration forces is shown in the α-helix to β-sheet conformational transition that accompanies the racemic self-assembly of polylysine [727].

Although the native state of a protein resides at a minimum on the potential energy surface, there is no reason to suppose that this structure is the global minimum free energy structure as its folding route is a guided, rather than random, process.

Compatible solutes (osmolytes, e.g. betaine), that stabilize the surface low-density water and increase the surface tension, will also stabilize the protein's structure (see also the Hofmeister effect and the solubility of non-polar gases).

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Denaturation

Water is critical, not only for the correct folding of proteins but also for the maintenance of this structure. The free energy change on folding or unfolding is due to the combined effects of both protein folding/unfolding and hydration changes. These compensate to such a large extent that the free energy of stability of a typical protein is only 40-90 kJ mol-1 (equivalent to very few hydrogen bonds), whereas the enthalpy change (and temperature times the entropy change) may be greater than ±500 kJ mol-1 different. There are both enthalpic and entropic contributions to this free energy that change with temperature and so give rise to heat denaturation and, in some cases, cold denaturation. The midpoint temperatures of both heat and cold denaturation may be determined from peaks in the temperature dependence of the heat capacity, where additional heat is being absorbed by the intermediate structures. Protein unfolding (denaturation) is easily understood but the widespread existence of protein unfolding at low temperatures is surprising, particularly as it is unexpectedly accompanied by a decrease in entropy [416].

The free energy on going from the native (N) state to the denatured (D) state is given by

. The overall

free energy change ( ) depends on the combined effects of the exposure of the interior polar and non-polar groups and their interaction with water together with the consequential changes in the water-water

interactions on , and

. The graph is meant to be indicative only. Denaturation is

only allowed when is negative; its rate is then dependent circumstances and may be fast or immeasurably slow.

The midpoint temperatures of both heat and cold denaturation may be determined from peaks in the temperature dependence of the heat capacity, where additional heat is being absorbed by the intermediate structures.

The enthalpy of transfer of polar groups from the protein interior into water is positive at low temperatures and negative at higher temperatures [150]. This is due to the polar groups creating their own ordered water, which generates a negative enthalpy change due to the increased molecular interactions. Balanced against this is the positive enthalpy change as the pre-existing water structure and the polar interactions within the protein both have to be broken. As water naturally has more structure at lower temperatures, the breakdown of the water structure makes a greater positive contribution to the overall enthalpy at lower temperatures.

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In contrast, the enthalpy of transfer of non-polar groups from the protein interior into water is negative below about 25°C and positive above [150]. At lower temperatures, non-polar groups enhance pre-existing order such as the clathrate-related ES structure [270], discussed elsewhere, generating enthalpy but this effect is lost with increasing temperature, as any pre-existing order is also lost. At higher temperatures, the creation of these clathrate structures requires an enthalpic input. Thus, there is an overall positive enthalpy of unfolding at higher temperatures. An equivalent but alternative way of describing this process is that at lower temperatures the clathrate-type structure optimizes multiple van der Waals molecular interactions whereas at higher temperatures such favorable structuring is no longer available.

At ambient temperature, the entropies of hydration of both non-polar and polar groups are negative [151] indicating that both create order in the aqueous environment. However these entropies differ with respect to how they change with increasing temperature. The entropy of hydration of non-polar groups increases through zero with increasing temperature, indicating that they are less able to order the water at higher temperatures and may, indeed, contribute to its disorder by interfering with the extent of the hydrogen-bonded network. Also, there is an entropy gain from the greater freedom of the non-polar groups when the protein is unfolded. In contrast, the entropy of hydration of polar groups decreases, becoming more negative with increasing temperature, as they are able to create ordered hydration shells even from the more disordered water that exists at higher temperatures.

Overall, protein stability depends on the balance between these enthalpic and entropic changes. For globular proteins, the ∆G of unfolding has a maximum 10-30°C, decreasing both colder and hotter through zero with the thermodynamic consequences of both cold and heat denaturation. The hydration of the internal polar groups is mainly responsible for cold denaturation as their energy of hydration is greatest when cold. Thus, it is the increased natural structuring of water at lower temperatures that causes cold destabilization of proteins in solution (i.e. the entropic cost of denaturation, due to the structuring of the water molecules around the exposed groups, is reduced). Heat denaturation is primarily due to the increased entropic effects of the non-polar residues (i.e. the increased entropy gain of the unfolded chain is not much reduced by the small amount of entropy loss caused to the solute). Although both processes have been reported to lead to irreversible changes, which often occur cooperatively, cold inactivation in supercooled water is usually likely to be reversible and it is any ice crystal formation that leads to observed irreversible effects.

Protein stability has been directly tied to the equilibrium structuring of water between low-density and higher density forms [210, 416] (see also). This provides an equivalent but alternative way of looking at the above analysis. Effectively the denaturation is treated as increased solubility of the unfolded form in a manner similar to that given in the treatment of the anomalous solubility behavior of non-polar gases.

a To avoid such activity loss, proteins generally avoid crystal formation, perhaps by evolutionary design involving surface kosmotropic lysine residues that minimize self-aggregation [771].

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b An approximate comparison of the hydrophobicity of the amino acids is given in the table opposite. Further discussion of relative hydrophobicities is given elsewhere as is a classification of hydrophobicity scales [633]. It should be noted that such hydrophobic interactions are particularly important in stabilizing interdomain and quaternary interactions. [Back]

Acids and Bases

Each protein has an optimum pH at which it is most stable, and in the case of an enzyme, where it has optimal activity. This optimum pH may vary from pH 1-2 (for an enzyme such as pepsin, which is active in the stomach) to pH 8-9 (for an enzyme such as lipase, which is present in the intestine). At the optimum pH, the protein has a complex network of positive and negative charges, necessary to maintain its structure and activity.

Addition of an acid or base to a solution of protein which is at its optimal pH, changes the number and distribution of charges on the protein. At low pH values, the net positive charge on a protein will increase, as carboxyl and amino groups are protonated, while at high pH values, the net negative charge will increase. These changes in the ionic interactions can destabilize or cause denaturation of the protein structure by:

o the development of like charges which cause internal repulsion o the loss of opposite charges which may have been involved in attractive forces o the loss of surface charges which may have kept individual protein molecules

apart

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If the addition of acid or base changes the pH to a point where there is no net charge on the protein, proteins may come together mainly through hydrophobic interactions, form agglomerates and precipitate from solution.The pH where there is no net charge is referred to as the Isoelectric point.

Isoelectric Point (pI)

The isoelectric point (or pI) of a protein is the pH at which the protein has an equal number of positive and negative charges. This attribute may be determined by isoelectric focusing (IEF), which is also the first dimension of 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis).

As a rule, isoelectric point is not a very good tool for identifying a protein. The experimental values determined by electrophoresis are not very accurate and all sorts of posttranslational modifications can produce a difference between experimental pI and theoretical pI (which the tools use) of as much as 2 whole numbers! Allowing that much error into the match makes it completely useless.

What might work is predicting isoelectric point values. There will certainly still be large differences but if you have an idea what your proteins are and are using a larger pH gradient for electrophoresis, it might help narrow the identity of spots and bands.

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[http://chimge.unil.ch/En/ph/1ph79.htm]

Amino acids, under their zwitterionic form behave as amphoteric particles; the pH of their solutions is given by :

This pH is called isoelectric pH because the zwitterion is overall neutral. It is noted pHi

Distribution diagram of the species

Electrophoresis of Proteins Proteins can be separated and purified. Methods for separating proteins take advantage of properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis of their binding properties. The source of a protein is generally tissue or microbial cells. The cell must be broken open and the protein must be released into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or to isolate organelles. Once the extract or organelle preparation is ready, a variety are available for separation of proteins. Ion-exchange chromatography can be used to separate proteins with different charges (similar to the way amino acids are separated). Other chromatographic methods take advantage of differences in size, binding affinity, and solubility. Nonchromatographic methods include the selective precipitation of proteins with salt, acid, or high temperatures. In addition to chromatography, another important set of methods is available for the separation of proteins, based on the migration of charged proteins in an electric field, a process called (gel) electrophoresis. Gel electrophoresis is especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or the degree of purity of a particular protein preparation.

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Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Amino acids differ not only in R-group characteristics but also in molecular weight. Different amino acids are linked together in a linear chain by peptide bonds in various combinations and sequences to form specific proteins. A protien may be comprised of amino acids from all of the categories. The net charge of a protein will depend on its amino acid composition. If it has more positively charged amino acids such that the sum of the positive charges exceeds the sum of the negative charges, the protein will have an overall positive charge and migrate to the cathode (negatively charged electrode) in an electrical field. Proteins even with a variation of one amino acids will have a different overall charge, and thus are electrophoretically distinguishable. If a protein is electrophoresed before the disulfide bonds are broken, it will yield only one band of 125,000 Daltons molecular weight on the gel. That is, the protein will move as one entity and the peptide chains cannot be distinguished. However, after treatment with a reducing agent, the protein will yield four distinct bands with a combined molecular weight of 125,000 Daltons--the same as the original protein. The gel results will show that some of the high molecular weight bands from samples not treated with the disulfide reducing agent are missing in the samples treated with the disulfide reducing agent. However, two or more bands are imaged on the gel lane containing the treated samples, thereby replacing each of the missing high molecular weight bands from the samples that were not treated. The additional bands that appear in the treated samples represent the individual polypeptides that make up complex proteins. The break up of complex proteins into their respective polypeptides allows us to study the structure of proteins that result from the interaction of several genes. A gene is a discrete unit of hereditary information that usually specifies a protein. A single gene provides the genetic code for only one polypeptide. Thus, a protein consisting of four polypeptides requires the interaction of four genes to synthesize that specific protein. A molecular weight protein marker is used to prepare a standard separation curve with which various unknown proteins or polypeptide fractions can be identified.

Protein Denaturation: Organic Solvents

Since nonpolar side chains are more soluble in organic solvents than in water, hydropobic interactions are weakened by organic solvents. On the other hand, since the stability and formation of peptide hydrogen bonds are enhanced in a low-permittivity environment, centain organic solvents may actually strengthen or promote formation of peptide hydrogen bonds. For example, 2-chloroethanol causes an increase in a-helix content in globular proteins. Organic solvents, such as acetone and alcohol, also can cause denaturation of proteins and their effects are reduced by low temperatures. The net effect of an organic solvent on protein structure, therefore, usually depends on the magnitude of its effect on various polar and nonpolar interactions. At low concentration, some organic solvents can stabilize several enzymes against

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denaturation. At high concentrations, however, all organic solvents cause denaturation of proteins because of their solubilizing effect on nonpolar side chains.

A study of the effect of alcohols was carried out on yeast glyceraldehyde phosphate dehydrogenase. Using different alcohols, it was found that the longer the aliphatic chain, the more denaturing the alcohol was (Fig. 4). The percentage of n-alcohol required to cause 50% denaturation in 30 min at 30oC decreased by a factor of exactly 2 for every methylene added to the alcohol.

[Fig. 4. Denaturation of yeast glyceraldehyde phosphate dehydrogenase by alcohols.] (Source: Scopes, RK. 1994. Protein Purification: Principles and Practice. 3rd ed. Springer-Verlag New York, Inc. USA. P. 95-101.)

Source: Fennema, OR. 1996. Food Chemistry. 3rd Edition. Marcel Dekker, Inc., New York. Chapter 6.

Scopes, RK. 1994. Protein Purification: Principles and Practice. 3rd ed. Springer-Verlag New York, Inc. USA. P. 95-101.)

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Protein Denaturation: Detergents

Detergents, such as sodium dodecyl sulfate (SDS), are powerful protein denaturing agents. The mechanism involves preferential binding of detergent to the denatured protein molecule. Detergent-induced denaturation is usually irreversible.

Synthetic detergents are among the most effective denaturing agents known. These compounds have the ability to form a chemical bridge between hydrophobic and hydrophilic environments, thus disrupting or diminishing the hydrophobic forces needed to maintain native protein structure.

Application

Polyacrylamide gel electrophoresis (PAGE) with an anionic detergent sodium dodecyl sulfate (SDS) is a form of denaturing electrophoresis used to separate protein subunits by size. Proteins are solubilized in a buffer containing SDS and a reducing agent mercaptoethanol or dithiothreitol, to dissociate the protein into subunits and reduce disulfide bonds. Proteins bind SDS, become negatively charged, and are separated based on size.

[Fig. 5 SDS-PAGE Example]

Sources: Fennema, OR. 1996. Food Chemistry. 3rd Edition. Marcel Dekker, Inc., New York. Chapter 6. Fennema, OR. 1976. Principles of Food Science. Marcel Dekker, Inc., New York. chapter 5.

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Protein Denaturation: Chaotropic Agents

Salts affect protein stability in two different ways. At low concentrations, ions interact with proteins via nonspecific electrostatic interactions. This electrostatic neutralization of protein charges usually stabilizes protein structure. However, at higher concentrations (> 1M), salts have ion specific effects that influence the structural stability of proteins. Salts such as Na2SO2 and NaF enhance, whereas NaSCN and NaClO4 weaken it. Protein structure is influenced more by anions than by cations. The relative ability of various anions at iso-ionic strength to influence the structural stability of protein (and DNA) in general follows the series

F-< SO42-<Cl-<Br-<I-<ClO4-< SCN-<Cl3CCOO-

This ranking is known as the Hofmeister series or chaotropic series.

Fluoride, chloride, and sulfate salts are structure stabilizers, whereas the salts of other anions are structure destabilizers.

Salts that stabilize proteins enhance hydration of proteins and bind weakly, whereas salts that destabilize proteins decrease protein hydration and bind strongly. In other words, the denaturing effect of chaotropic salts might be related to destabilization of hydrophobic interactions in proteins.

High concentrations (6-8M) of compounds that tend to break hydrogen bonds, such as urea and guanidine salts, also cause denaturation of proteins. These substances apparently disrupt hydrogen bonds which hold the protein in its unique structure. However, there also is evidence suggesting that urea and guanidine hydrocholoride may disrupt hydrophobic interactions by promoting the solubility of hydrophobic residues in aqueous solutions.

"Salting out"& its applications

If the concentration of neutral salts is at a high level (>0.1M), in many instances the protein precipitates. This phenomenon apparently results because the excess ions (not bound to the protein) compete with proteins for the solvent. The decrease in solvation and neutralization of the repulsive forces allows the proteins to aggregate and precipitate. This effect is called "salting out".

The inorganic salting out process has wide-spread application as a downstream processing for proteins both at laboratory and industrial scales. Examples include recovery of proteins such as diagnostic enzymes, hormones, pharmaceuticals and food proteins. Salting-out by ammonium sulphate is a typical protein separation technique whereby a concentrated salt is added to a protein solution producing a protein-rich precipitate.

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Sources: Fennema, OR. 1996. Food Chemistry. 3rd Edition. Marcel Dekker, Inc., New York. Chapter 6. Fennema, OR. 1976. Principles of Food Science. Marcel Dekker, Inc., New York. chapter 5. Shih, YC. Prausnitz, JM. and Blanch, HW. 1992. Some characteristics of protein precipitation by salts. Biotechnology and Bioengineering 40(10):1155-1164.

Kosmotropes and Chaotropes

The terms 'kosmotrope' (order-maker) and 'chaotrope' (disorder-maker) originally denoted solutes that stabilized, or destabilized respectively, proteins and membranes. Later they referred to the apparently correlating property of increasing, or decreasing respectively, the structuring of water. Although useful, the terminology may sometimes be misleading as such properties may vary dependent on the circumstances, method of determination or the solvation shell(s) investigated. For example a solute may not always act in the same way at different concentrations or in the presence of macromolecules or gels [276]. Also some solutes with less well-defined properties (e.g. urea) are sometimes classified as kosmotropes [276] and sometimes as chaotropes [283]. An alternative term used for kosmotrope is 'compensatory solute' as they have been found to compensate for the deleterious effects of high salt contents (which destroy the natural hydrogen bonded network of water) in osmotically stressed cells, but again behavior as a kosmotrope in one system does not mean that a solute may behave as a 'compensatory solute' in another or even that it will stabilize the structuring of water in a third. Both the extent and strength of hydrogen bonding may be changed independently by the solute but either of these may be, and has been, used as measures of order making. It is, however, the effects on the extent of quality hydrogen bonding that is of overriding importance as true kosmotropes shift the local less dense water (e.g. ES) more dense water (e.g. CS) equilibrium to the left and chaotropes shift it to the right. The ordering effects of kosmotropes may be confused by their diffusional rotation, which creates more extensive disorganized junction zones of greater disorder with the surrounding bulk water than less hydrated chaotropes. It seems clear that most kosmotropes do not cause a large scale net structuring in water [595].

Ionic kosmotropes should be treated differently from non-ionic kosmotropes, due mainly to the directed and polarized arrangements of the surrounding water molecules. Generally, ionic behavior parallels the Hofmeister series. Large singly charged ions, with low charge density (e.g. SCN-, H2PO4

-, HSO4-, HCO3

-, I-, Cl-, NO3-, NH4

+, Cs+, K+, (NH2)3C+ (guanidinium) and (CH3)4N+ (tetramethylammonium) ions; exhibiting weaker interactions with water than water with itself and thus interfering little in the hydrogen bonding of the surrounding water), are chaotropes whereas small or multiply-charged ions, with high charge density, are kosmotropes (e.g. SO4

2-, HPO42-, Mg2+, Ca2+, Li+, Na+, H+, OH- and HPO4

2-, exhibiting stronger interactions with water molecules than water with itself and therefore capable of breaking water-water hydrogen bonds).a The radii of singly charged chaotropic ions are greater than 1.06Å for cations and greater than 1.78Å for anions [284]. Thus the hydrogen bonding between water molecules is more broken in the immediate vicinity of ionic kosmotropes than ionic chaotropes. Reinforcing this conclusion, a Raman spectroscopic study of the hydrogen-bonded structure of water around

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the halide ions F-, Cl-, Br- and I- indicates that the total extent of aqueous hydrogen bonding increases with increasing ionic size [685]. It is not unreasonable that a solute may strengthen some of the hydrogen bonds surrounding it (structure making; e.g. kosmotropic cations will strengthen the hydrogen bonds donated by the inner shell water molecules) whilst at the same time breaking some other hydrogen bonds (structure breaker; e.g. kosmotropic cations will weaken the hydrogen bonds accepted by the inner shell water molecules) [274], so adding to the confusion in nomenclature. Other factors being equal, water molecules are held more strongly by molecules with a net charge than by molecules with no net charge; as shown by the difference between zwitterionic and cationic amino acids [532].

Weakly hydrated ions (chaotropes, K+, Rb+, Cs+, Br-, I-, guanidinium+) may be 'pushed' onto weakly hydrated surfaces by strong water-water interactions with the transition from strong ionic hydration to weak ionic hydration occurring where the strength of the ion-water hydration approximately equals the strength of water-water interactions in bulk solution (with Na+ being borderline on the strong side and Cl- being borderline on the weak side) [284]. Neutron diffraction studies on two important chaotropes (guanidinium and thiocyanate ions) show their very poor hydration, supporting the suggestion that they preferentially interact with the protein rather than the water [488]. In contract to the kosmotropes, there is little significant difference between the properties of ionic and nonionc chaotropes due to the low charge density of the former.

Optimum stabilization of biological macromolecule by salt requires a mixture of a kosmotropic anion with a chaotropic cation. As ionic kosmotropes primarily achieve their increased structuring solely within their hydration shell, they partition into the more dense (CS) water where they can obtain this hydration water more readily, whereas the ionic chaotropes, by avoiding interference with water's hydrogen-bonded network, tend to clathrate formation within the less dense (ES) environment. Thus there is agreement with the defining characteristic of an ionic chaotrope in that it partitions selectively into low-density water whereas a kosmotrope partitions selectively into high-density water [276]. The stabilizing of structured low-density water in turn stabilizes hydrophobic interactions.

Non-ionic kosmotropes are very soluble well-hydrated molecules with little tendency to aggregate, having no net charge and strongly hydrogen bonding to water, that stabilize the structure of macromolecules in solution. They are preferentially solubilized within the bulk of the solution and excluded from the solvation layers of macromolecular surfaces. Consequentially, they decrease the water diffusion around the proteins and the exchange rate of backbone amide protons [621]. This leads to the dehydration of such surfaces and ensures that they are less flexible and therefore more thermally stable but less enzymatically active.b Kosmotropes reduce the volume of water available to hydrate the larger surface exposed by denatured proteins, so tending to prevent the denaturation process (the 'excluded volume' effect).These kosmotropes may be divided into two groups which act somewhat differently: (1) polyhydroxy compounds fit in well with the hydrogen bonding arrangements, but stabilize any cluster 'flickering' via hydrogen bond rearrangement, as their hydroxyl groups are similarly separated to water-water separations (see sugar hydration), and (2) zwitterions where the balance between hydrophilicity, hydrophobicity, anionic and cationic characteristics ensures good solubility but only weak net interactions to surrounding water.

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Chaotropes break down the hydrogen-bonded network of water, so allowing macromolecules more structural freedom and encouraging protein extension and denaturation. Kosmotropes are stabilizing solutes which increase the order of water (such as polyhydric alcohols [307], trehalose, trimethylamine N-oxide, glycine betaine, ectoine, proline and various other zwitterions) whereas chaotropes create weaker hydrogen bonding, decreasing the order of water, increasing its surface tension (but see anomaly) and destabilizing macromolecular structures (such as guanidinium chloride and urea at high concentrations). Recent work has shown that urea weakens both hydrogen bonding and hydrophobic interactions but glucose acts as a kosmotrope, enhancing these properties [283]. The idiosyncratic behavior of urea may well be due to its concentration-dependent oligomerization; cyclic hydrogen-bonding dimers and oligomers behaving differently from monomers [364]. Thus, when urea molecules are less than optimally hydrated (about 6 - 8 moles water per mole urea) urea hydrogen bonds to itself and the protein (significantly involving the peptide links [528]) in the absence of sufficient water, so becoming more hydrophobic and hence more able to interact with further sites on the protein, leading to localized dehydration-led denaturation. Guanidinium is a planar ion that may form weak hydrogen bonds around its edge but possesses rather hydrophobic surfaces that may interact with similar protein surfaces to enable protein denaturation [571]. Both denaturants may cause protein swelling and destructuring by sliding between hydrophobic sites and consequently dragging in hydrogen-bound water to complete the denaturation.

Rather unexpectedly, whereas L-amino acids and D-glucose prefer a less dense (ES) environment, D-amino acids and L-glucose prefer more dense (CS) water [574]; a consequence of which may be that condensed polymers of the former (rather than their optical isomers) may have formed in primordial clays so setting the trend for the molecular evolution [374]. Although the arrangement of oxygen atoms within water clusters may be symmetrical, the energetic preference for particular hydrogen-bonding arrangements ensures the necessary chirality.

Kosmotropes

a Trimethylamine N-oxide

b Proline

c Ectoine; R varying

d α,α-Trehalose

e Glycine betaine

f 3-Dimethylsulfoniopropionate

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Generally the kosmotropic/chaotropic nature of a solute is determined from the physical bulk properties of water, often at necessarily high concentration. The change in the degree of structuring may be found, for example, using NMR ([307]) or vibrational spectroscopy. Protein-stabilizing solutes (kosmotropes) increase the extent of hydrogen bonding (reducing the proton and 17O spin-lattice relaxation times) whereas the NMR chemical shift may increase (showing weaker bonding e.g. the zwitterionic kosmotrope, trimethylamine N-oxide) or decrease (showing stronger bonding e.g. the polyhydroxy kosmotrope, trehalose). Trehalose shows both a reduction in chemical shift and relaxation time, as to a lesser extent does the protein stabilizer (NH4)2SO4, whereas NaCl only shows a reduction in chemical shift and the protein destabilizer KSCN shows an increase in relaxation time and a reduction in chemical shift (note these NMR parameters are both time and structurally averaged values, where the weighting of the averaging is unclear) [281]. Vibrational spectroscopy may make use of the near-IR wavelength near 5200 cm-1 (v2 + v3 combination), which shifts towards longer wavelength (smaller wavenumber) when hydrogen bonds are stronger [282]. It should be noted however that ranking of kosmotropic/chaotropic character by different measures show inconsistencies.

One of the most important kosmotropes is the non-reducing sugar α,α-trehalose. It should perhaps be noted that trehalose has a much more static structure than the reducing sugars, due to its lack of mutarotation, or the other common non-reducing disaccharide, sucrose, due to its lack of a furan ring. Both Inelastic Neutron Scattering [543] and 17O-NMR show that a rotational restriction exists in trehalose-bound water, indicating that trehalose is definitely a net water-structurer. Trehalose interferes with the tetrahedral network of water, structuring out to at least the third solvation layer, in such a way as to reduce the amount of freezable water [285]. This is consistent with an increase in the local structuring, due in part to maximizing the number of singly hydrogen-bonded water molecules (to trehalose) without any (trehalose) intramolecular hydrogen bonds or oriented water molecules held by two (trehalose) hydrogen bonds. There is also reduced terahedrality amongst the associated water molecules [660]. At lower water contents, essentially all the water is associated with single hydrogen bonds to trehalose or first shell water with consequential reduction in the ability to form four hydrogen bonds per water molecule but retaining an expanded structure. Trehalose is particularly effective at stabilizing macromolecules as it has a large hydrated volume (2.5 times that of sucrose, indicating that there is less water for the same volume; i.e. low-density water is present) [279]. Trehalose can also protect membranes in anhydrobiosis by hydrogen bonding directly to the phosphate groups in the phospholipids at low water content, so spreading their head groups and reducing the membrane's tendency to undergo phase transitions and thus leak during rehydration [308]. As a further protective action trehalose crystals, formed at low water content, can lose (and regain) up to two molecules of water per molecule without changing their crystal structure or volume [327].

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a Opposite is shown a cartoon illustrating the main difference between ionic chaotropes and kosmotropes. Water molecules surrounding ionic chaotropes tend towards a convex dodecahedral (clathrate) arrangement, whereas those surrounding ionic kosmotropes tend towards a puckered arrangement with a number of water molecules lying close to the ion (see elsewhere for more details), causing bent and broken hydrogen bonding further out. The dodecahedral water cluster may be thought to constantly interconvert between convex and puckered forms with the preferred clustering governed by the chaotropic-kosmotropic balance. [Back]

The water dodecahedra shown only as connected O-atoms of water, surrounding the central ion..

b A few adventitious and deleterious enzymatic and non-enzymatic reactions may occur during lengthy dehydration, which may be reduced by the presence of specific amphiphilic molecules that concentrate at the interfaces [321]. [Back]

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