Protein denaturation

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Protein denaturation

Protein denaturation involves destruction of secondary and tertiary structures while the primary structure remains the same.

It is the reverse of protein folding, producing order in the form of those structures

Denaturation of proteins changes the functional properties in terms of effects on solution

Denaturation effects

Denaturation effects

Description

1. Denaturation of globular

proteins decreases solubility

• Globular proteins have most of its hydrophobic amino acids located in the core of the protein while most of its charged, polar groups face the water molecules.

• When denaturation occurs, hydrophobic amino acids are exposed, decreasing the number of protein-water interactions, hence decreasing solubility.

2. Loss of biological activity

• Enzymes have specific active site owing to

their tertiary structure.

• Denaturation causes loss of tertiary structure, resulting in loss of their biological activity.

3. Increases susceptibility to

proteolytic attack

• Proteases bind to specific aa residues of protein to cleave peptide bonds. • Denaturation exposes aa sequence of

protein for proteases binding.

Events occurring during protein denaturation

❖ Water entropy

• Protein denaturation decreases water entropy

When proteins are denatured, hydrophobic amino acids are exposed to water which in turn form clathrate around them. Clathrate formation decreases water mobility, hence decrease entropy of water.

• 2nd law of thermodynamic: Close system always try to increase entropy with time

There is a thermodynamic drive for non-polar aa to associate with each other so as to increase entropy of the system. Thus, protein folds, increasing degree of water disorder

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• Stability of folded state

Once folded, non-covalent interactions between protein groups contribute to its stability

NOTE and Summary:

1. Increase in water entropy is the driving force for hydrophobic interactions

2. The higher the temperature, the higher is water entropy, thus stronger hydrophobic interactions 3. Protein stability is related to the equilibrium structure of water

❖ Chain entropy

Protein denaturation increases chain entropy by 2 means:

1. Increased atomic motions in relaxed chain

The atomic bonds that make the polypeptide chains (C-H, O-H etc) can vibrate and rotate. These motions are restricted when protein is folded, unlike in unfolded state.

2. Refolding

Refolding is a complex process and can go through large number of folding forms, increasing chain entropy.

Effects of temperature on Protein stability

An increase in temperature results in:

1. Weaker electrostatic interactions as molecules gain K.E

2. Stronger hydrophobic interactions since water entropy is increased (which is the driving force for protein folding)

3. Greater chain entropy as atomic and molecular motions are increased

Why are hydrophobic interactions endothermic?

When 2 hydrophobic molecules associate with each other, hydrogen bonds between water clathrate and each molecule are broken, increasing enthalpy (H) (bonds broken = + E absorbed)

Denaturation temperature TD

TD is the temperature at which the sum of all free energy contributions equal to 0

Opposing denaturation: An increase in temp results in an increase in water entropy, opposing protein denaturation (or favours protein folding as hydrophobic interactions are made stronger)

Favouring denaturation: With increasing temp, chain entropy increases (higher K.E, more atomic and

molecular motions), favouring denaturation

These 2 effects balance out until a certain temp – called denaturation temperature – is reached where solvent/water entropy starts to decrease due to new protein-water interactions and chain entropy is greater. This promotes denaturation.

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➢ Denaturation temperature occurs when solvent entropy decreases after it reaches its maximum point while chain entropy keeps increasing.

NOTE: Denaturationpromotes aggregation because:

1. Decrease protein solubility due to increase surface hydrophobicity

2. Exposed hydrophobic patches tend to associate together to increase water entropy

Physical Functional properties of proteins

1. Viscosity modulation

2. Gelation

3. Interfacial properties (surface tension, emulsification) 4. Solubility measurement

5. Water holding 6. Heat stability

❖ Viscosity modulation

Definition: It is a measure of the resistance of a fluid which is deformed by force (sheer stress)

Importance: It affects consistency of foods such as yoghurt, milk shake, tomato sauce; it affects processing characteristics during pumping, heating, cooling, spray drying – for ex in manufacture of milk powder.

Factors affecting viscosity of

protein solution

Description

1.

Protein intrinsic

properties

a. Molecular weight

Protein itself contributes to viscosity of solution. Large proteins have larger molecular dimension (size and space it occupies) than smaller ones, thus increasing solution viscosity for the same amount of protein.

b. Solution structure

Proteins of same molecular mass but those occupying larger volume in solution produce higher solution viscosity as it decreases the ease of flow of solution.

c. Protein charge

The number of charged amino acids affects water binding. ➢ Globular protein.

2. Protein-solvent

interactions

• Restricting water movement increases viscosity. It is mainly affected by pH of solution and ionic strength. • pH affects charge on protein surface

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3. Protein-protein

interactions

(intermolecular forces)

• Partial protein network structure that immobilises water increases viscosity.

• It is affected by solution pH and ionic strength.

Some proteins exhibit shear thinning behaviour, i.e viscosity decreases when flow rate increases. This

occurs with highly solvated protein systems where the high shear rates remove the solvated layers, increasing solution flow. This causes a decrease in frictional resistance of protein solution due to

orientation of protein molecules in the direction of flow. Protein hydration sphere is deformed in direction of facilitating flow. Globular protein shows this feature. Low shear rates have little effect of viscosity.

Irreversible viscosity

Viscosity cannot be recovered once flow is stopped. These proteins cannot re-associate themselves in the solution. Fibrous proteins like gelatine (collagen) and myosin (muscle) display this feature.

Reversible (Thixotropic System)

Viscosity can be recovered once flow is stopped. Globular proteins like soy and whey proteins are

examples of proteins that have a thixotropic system. Such food system includes tomato sauce, BBQ sauce where whey protein is added to these sauces.

Gelation

Polymer hydrodynamics

Description

1. Dilute Regime

It occurs in dilute protein solutions where each protein molecule is independent of each other. They occupy a hydrodynamic space in the liquid (dotted line).

2. Semi-dilute Regime

Proteins are very crowded and occupies the full space. This results in rapid increase in viscosity due to the protein-protein interactions – close

proximity. This may occur at relatively low concentration for large protein molecules.

3. Concentrated Regime

There is loss of protein integrity as they are totally

or partially denatured and intermingle with each

other. They are fully overlapped. The solution properties are influenced by protein-protein interactions (protein chains) at this concentration.

What are gels?

Gel are semi solid structures that are formed when a viscous fluid is changed into a 3D network with

viscoelastic characteristics – when force is applied, it will not change shape/deform.

Gels are formed by protein-protein chains interaction (between different polypeptides or polymers of sugar) that form a 3D protein network that can hold and trap water molecules.

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Steps involved in making gel:

1. Change in conformation

There is the need for a change in conformation, often heat-induced, or partial denaturation of protein molecules which lead to increase in viscosity due to an increase in molecular dimensions of unfolding proteins and an increase in protein-water interactions.

SUMMARY: Change in protein conformation → increase molecular dimensions of unfolding protein + increase in protein-water interactions → increase viscosity

2. Aggregation

There will be gradual association/aggregation of individual denatured proteins (aggregation does NOT occur along the entire length of polypeptides but only on some sections). This produces a 3D structure in the solution, leading to an exponential increase in viscosity as material approaches a continuous network. NOTE: Step 1 and 2 are important. If step 2 is too fast compared to step 1, we will have a random network that does not have a firm structure where instead of trapping water, it will leak and be lost. Syneresis occurs.

➢ A random network unable to trap water is called syneresis.

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Carbohydrates

Isomerism

Types of isomers

Example

1.

Structural

isomer

They are compounds with the same molecular formula but have different bonding arrangement.

Ex: Glc and Fru have same empirical formula C6H12O6 but different

atoms arrangement.

2.

Stereoisomer

They have same sequence of bonded atoms but have different spatial arrangements

a.

Distereoisomers

Distereoisomers are non-mirror images, non-superimposable and all are hexoses.

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b. Enantiomers

Non superimposable mirror image of D and L-sugars

c. Epimers

Diastereoisomeric monossacharides that have opposite configurations of

1 hydroxyl group at only one position.

D-glucose & D mannose are epimers at C2 D-glucose & D-galactose are epimers at C4

d. Anomers

A pair of monosaccharide that differs only at position of C1-OH in ring form

Ex: α-D-Glucose or β-D-Glucose

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Similarities and Differences between caramelisation and Maillard Reaction

Similarities:

1. Both reactions involve sugars.

Caramelisation usually involves sucrose breakdown into glucose and fructose while Maillard reaction involves reducing sugars such as glucose

2. Both require heat for desirable reactions

3. Both reactions are favoured when water is limited 4. Both generate flavour compounds

In caramelisation, monosaccharides undergo decomposition to produce non-sugar molecules that provide flavours and aromas. In Maillard reaction, there is condensation and polymerisation of furfurals and hydroxymethylfurfurals (HMF) which produce melanoidins and aldehydes which provide flavours.

5. Both generate coloured compounds

Caramelisation involves dehydration of isosaccharan which in turn assemble to form coloured products such as caramelan, caramelin and caramelen. In Maillard, the production of brown pigments melanoidins provide colour.

6. Hydroxymethyl furfuryl (HMF) is a common end product

Differences

Caramelisation

Maillard Reaction

Involves decomposition, dehydration, dimerization, self-assembly reactions

Reaction occurs between amine and aldehyde or ketones

Reactions occur between sugar entities Reactions occur between sugars and amino acids High heat is necessary to obtain desirable reactions

(> 150 degree)

Medium heat is enough for aldehydes and ketones and amine to react together

Colours are formed due to the formation of several coloured compounds altogether

Colours are formed by polymers which are melanoidin only

Generates sugar derived low molecular weight compounds

Generates heterocyclic and sugar derived LMW compounds

No known toxic effects on health Heterocyclic compounds are carcinogenic

Similarities/Differences between enzymatic browning and Maillard Reactions

Similarities:

1. Both enzymatic browning and Maillard reactions involve amino acids and proteins 2. There is accumulation of nitrogen containing brown polymers

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Differences:

Enzymatic browning

Maillard Reaction

Does not involve sugars Involves reducing sugar and amino acids Reaction driven by enzyme actions (polyphenol

oxidase)

Chemical reactions which do not involve enzyme

Occurs at room temperature Requires high heat

Formation of orthoquinone intermediates Formation of amino containing and non-amino containing intermediates

Can be supressed by reducing agents Cannot be supressed by reducing agents Do not cause accumulation of large number of

small molecules

Accumulation of large number of small molecules such as furfural and HMF.

[PREVIEW FOR CARBOHYDRATES STOPS HERE]

Polysaccharides

Starch retrogradation

Starch consists of 2 polymers of amylose and amylopectin which are closely packed together to form granules.

NOTE: Undamaged starch granules are insoluble in cold water due to the collective strength of hydrogen bonds binding the chains together.

1. Gelatinisation

a) In cold water, starch granules are insoluble due to their strong H-bonds among the chains. b) As temperature is increased, there is initial gelatinisation. Water begins to be imbibed.

c) Heating release some of the amylose into solution to increase water interaction, hence increase solution viscosity.

d) Temperature is essential to entangle amylose and amylopectin bonded by H-bonds.

➢ Heat → amylose released → more water interaction → increase solution viscosity → gelation

2. Swelling

a) Water is imbibed between amylose and amylopectin, causing loosening of granule structure. Amylose is also released into solution.

b) This results in leaching of polymers and increases water binding capacity, hence increase viscosity.

3. Retrogradation

If heating is maintained together with stirring, the viscosity will eventually cease as the structure of the granules is damaged.

NOTE: If allow to cool, this will promote retrogradation. When solution is cooled, the free amylose in

solution start to recoil into its initial helical structure which can then re-associate with each other, forming amylose-amylose interaction through H-bonds. This leads to precipitation of…

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References