PROTEINS

• SOLUBILITY

• VISCOSITY

BY:KRITI RAWAL

PROTEIN SOLUBILITY

WHAT DO WE MEAN BY SOLUBILITY OF A PROTEIN?

IT IS THE THERMODYNAMIC MANIFESTATION OF THE EQUILIBRIUM BETWEEN protein-protein AND protein-solvent INTERACTIONS.

It is represented as :

Protein-protein

solvent-solvent

Protein-solvent

• The solubility of proteins in aqueous buffers depends on the distribution of hydrophilic and hydrophobic amino acid residues on the protein’s surface.

• Hydrophobic residues predominantly occur in the globular protein core, but some exist in patches on the surface.

• Proteins that have high hydrophobic amino acid content on the surface have low solubility in an aqueous solvent.

• Charged and polar surface residues interact with ionic groups in the solvent and increase the solubility of a protein.

INTERACTIONS INFLUENCING PROTEIN SOLUBILITY

HYDROPHOBIC

PROMOTE PROTEIN-PROTEIN

INTERACTIONS

DECREASE SOLUBILITY

IONIC

PROMOTE PROTEIN-WATER INTERACTIONS

INCREASE SOLUBILITY BY 2 WAYS:

1) ELECTROSTATIC REPULSION BETWEEN PROTEIN MOLECULES

2) REPULSION BETWEEN HYDRATION SHELLS

AROUND IONIC GROUPS

BIGELOW’S THEORY ABOUT SOLUBILITY OF PROTEINSolubility of a protein is related to:

• AVERAGE HYDROPHOBICITY OF AMINO ACID RESIDUES

Δg=ΣΔgresidue /n

Where,Δgresidue = hydrophobicity of amino acid residuen = total number of residues in the protein

• CHARGE FREQUENCY

σ=(n++n-)/nWhere,

σ = charge frequency

n+ = total number of positively charged residues in the proteinn- = total number of negatively charged residues in the protein

CLASSIFICATION OF PROTEINS ON THE BASIS OF SOLUBILITY :

ALBUMINS GLOBULINS GLUTELINS PROLAMINES

• Soluble in water at ph=6.6

• Ex: serum albumin, ovalbumin, alpha-lactalbumin

• Soluble in dilute salt solutions at ph=7.0

• Ex: beta-lactoglobulin

• Soluble only in acid(ph=2) and alkaline(ph=12) solutions

• Ex: wheat glutelins

• Soluble in 70% ethanol

• Ex: zein,gliadins

HYDROPHILIC HYDROPHOBIC

FACTORS AFFECTING PROTEIN SOLUBILITY

1. pH• pH<pI : proteins carry net positive charge (soluble)• pH>pI : proteins carry net negative charge (soluble)• pH=pI : positive and negative charges balanced ,minimum

electrostatic repulsion,aggregation and precipitation via hydrophobic interactions (minimum solubility)

Exceptions:• Beta-lactoglobulin (pI=5.2) and BSA (pI=5.3) are highly soluble

at their pI because of large ratio of surface hydrophilic groups to non polar groups

Alterations:• Heat denaturation alters pH-solubility profile of proteinsEx: Native whey protein isolate is soluble between pH=2-9 but heating at 70⁰C/1-10min causes solubility to decrease with minimum solubility at pH=4.5 due to increase in hydrophobicity of the protein surface because of unfolding

2. IONIC STRENGTH• Ionic strength (IS) of a salt solution is given by :

µ=0.5ΣCiZi2

Where,Ci=Concentration of ionZi=Valency of ion

• At IS<0.5,ions neutralize charges at the surface of protein.• Solubility decreases for proteins with more non polar patches

(soy protein) and increases for proteins with polar patches (beta-lactoglobulin).

• As salt concentration is increased upto 1,salts have ion specific effects on protein solubility. Sulphate and fluoride salts progressively decrease solubility (salting out) whereas thiocyanate and perchlorate salts increase solubility(salting in).

HOFMEISTER SERIES

• At constant µ, anions promoting solubility are in increasing order : SO4

2-<F-<Cl-<Br-<I-<ClO4--<SCN-

• At constant µ, anions promoting solubility are in increasing order :Ca2+<Mg2+<Li+<Na+<K+<NH4+

3. TEMPERATURE• At constant pH and ionic strength, solubility increases

within temperature range (0⁰C-40⁰C).• Above 40 ⁰C, increase in thermal kinetic energy causes

protein denaturation(unfolding),exposure of non polar groups, aggregation and precipitation leading to decrease in solubility.

Exceptions :• Highly hydrophobic proteins like beta-casein and some

cereal proteins

4. ORGANIC SOLVENTS• Addition of organic solvents,like ethanol and acetone

lower the permittivity of an aqueous medium. This increases intra and inter molecular electrostatic forces.

• Repulsive intramolecular and intermolecular forces cause unfolding of the protein molecule and favour intermolecular hydrogen bonding between exposed peptide groups, leading to precipitation of protein and reduced solubility.

SIGNIFICANCE OF PROTEIN SOLUBILITY

• Used as a measure of the extent of denaturation of protein during extraction, isolation and purification processes (since solubility is related to structural states).

• Index of potential applications of proteins.

• Solubility characteristics are represented by protein solubility index (PSI) or protein dispersibility index (PDI). PSI of commercial protein isolate ranges from 25-80%.

PROTEIN VISCOSITY

WHAT DO WE MEAN BY VISCOSITY?

IT IS THE RESISTANCE TO FLOW UNDER AN APPLIED FORCE (SHEAR STRESS)

For an ideal solution, shear stress (force per unit area) is directly proportional to shear rate (velocity gradient between layers of the liquid, dv/dr).

It is expressed as :

F/A=η(dv/dr)

Where,η=viscosity coefficient

Fluids that obey this expression are called “NEWTONIAN FLUIDS”

Most macromolecular solutions like protein solutions do not display Newtonian behaviour especiallly at high protein concentrations. For such systems, η decreases when shear rate increases. This behaviour is known as :

PSEUDOPLASTIC OR SHEAR THINNING

and follows the relationship:

F/A=m(dv/dr)n

Where,m= consistency coefficientN=flow behaviour indexPseudoplastic behaviour arises

because of :• The tendency of protein molecules to orient in the direction of flow • Dissociation of weakly held dimers and oligomers into monomers

also contributes to shear thinning.

When shearing is stopped, viscosity may or may not return to the original value,depending upon the relaxation of protein molecules to random orientation.

• If protein molecules remain oriented,they do not gain original viscosity (ex: solutions of fibrous proteins like gelatin and actomyosin)

• If protein molecules get randomly oriented,they regain their original viscosity when flow is stopped (ex: solutions of globular proteins like soy,whey proteins). Such solutions are

THIXOTROPIC

Viscosity coefficient of protein solutions follows an exponential relationship with protein concentration because of :• Protein-protein interactions• Interactions between hydration spheres of protein molecules

At high protein concentrations or in protein gels,where protein protein interactions are numerous and strong, proteins display viscoelastic behaviour. Here, a specific amount of force called “YIELD STRESS” is required to initiate flow.

Viscosity behaviour of proteins depends on:

• Size • Shape • Protein-solvent interactions• Hydrodynamic volume (volume of hydrated molecules)• Molecular flexibility in hydrated state

The dependence of viscosity on shape and size of protein molecules follows the relationship:

ηsp=βC(v2+δ1v1)

Where,ηsp=specific viscosityβ =shape factorC=concentrationv1=specific volume of solventv2=specific volume of unhydrated protein (directly proportional to molecular flexibility)δ1=grams of water bound per gram of protein

Viscosity of dilute protein solutions can be expressed as :

• RELATIVE VISCOSITY (ηrel)

1. Ratio of viscosity of protein solution to that of the solvent.2. Its measured in an Ostwald capillary viscometer.3. Its expressed as:

ηrel = η/ ηo = ρt/ ρoto

Where,

ρ=density of protein solutionρo=density of solventt=time of flow of protein solution through the capillaryto=time of flow of solvent through the capillary

• SPECIFIC VISCOSITY (ηsp)

(The relative viscosity of a protein solution of known concentration minus 1. Its usually determined at low concentration of the protein)

ηsp=ηrel –1

• Reduced viscosity (The specific viscosity (ηsp) divided by the concentration, expressed in g/ml. Also known as VISCOSITY NUMBER. )

ηred= ηsp/C where, C= protein concentration

• INTRINSIC VISCOSITY ([η])

(The ratio of a solution’s specific viscosity to the concentration of the solute, extrapolated to zero concentration. It’s a measure of a protein's contribution to the viscosity of a solution.)

[η]=Lim ηsp/C

• http://link.springer.com/chapter/10• Food chemistry by Owen R Fenemma• http://www.bio-rad.com/LifeScience/pdf/

important_factors.pdf• Guide to Protein Purification edited by

Murray P. Deutscher• Protein Purification: Principles and Practice

by Robert K. Scopes

REFERENCES