Download PROTEIN ANALYSIS

PROTEIN
ANALYSIS
Introduction
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Proteins are polymers of amino acids
Major structural components of many
natural foods
Food analysts are interested in knowing the
total concentration, type, molecular
structure and functional properties of
proteins in foods.
Determination of Protein
Concentration
1. Kjeldahl Method
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A food digested with strong acid so that it
releases nitrogen which can be determined by a
suitable titration techniques
Does not measure the protein content directly
Conversion factor is needed to convert the
measured nitrogen concentration to a protein
concentration
Principles
A. Digestion
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Sample is heated in the presence of sulfuric
acid (oxidizing agent), anhydrous sodium
sulfate, and a catalyst.
Digestion converts any nitrogen in the food into
ammonia and other organic matter to CO2 and
H2O
Ammonia gas remains in acid solution because
it is in the form of ammonium ion which binds
to the sulfate ion to form ammonium sulfate
B. Neutralization
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The solution in the digestion flask is then made
alkaline by addition of sodium hydroxide which
converts the ammonium sulfate into ammonia
gas
The ammonia gas moves out into receiving flask
which contains an excess of boric acid
In receiving flask, ammonia gas converts to
ammonium ion and boric acid to borate ion.
C. Titration
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Titrate with standard sulfuric or hydrochloric acid
using a suitable indicator
The concentration of hydrogen ions required to
reach the end-point is equivalent to the
concentration of nitrogen
Once nitrogen content has been determined, it is
converted to a protein content using appropriate
conversion factor.
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Advantages:
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universal
high precision
good
reproducibility
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Disadvantages:
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does not give a measure
of true protein
different proteins need
different correction factors
the use of concentrated
sulfuric acid at high
temperature
time consuming
2. Enhanced Dumas Method
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Principles: a sample of known mass is combusted
in a high temperature to release CO2, H2O and
N2
Nitrogen content is measured by separating N2
from CO2 and H2O using a column
Also used conversion factor to determine protein
content
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Advantages
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Faster
Doesn’t need toxic
chemicals or catalysts
Easy to use
Samples can be
measured
automatically
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Disadvantages
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High initial cost
Does not give a
measure of true
protein
Small sample size
make its difficult to
obtain a
representative sample
3. Methods using UV-Visible Spectroscopy
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Use either the natural ability of proteins to absorb (or
scatter) light or chemically or physically modify
proteins to make them absorb (or scatter) light in the
region.
Calibration curve of absorbance versus protein
concentration must be built first
Main difference: the chemical groups which are
responsible for the absorption or scattering of
radiation
Direct Measurement at 280 nm
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Principle: tryptophan and tyrosine absorb ultraviolet
light strongly at 280 nm
Use the same w/length to measure protein
concentration
Advantages: simple to carry out, non-destructive and
no special reagents are required
Disadvantages: nucleic acid absorb strongly at 280nm
To overcome: measure the absorbance at two
different w/lengths
Biuret Method
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A violet-purplish color is produced when cupric
ions (Cu2+) interact with peptide bonds under
alkaline conditions
Absorbance is read at 540 nm
Advantages: no interference from materials that
absorb at lower w/lengths and less sensitive to
protein type
Disadvantages: low sensitivity
Lowry Method
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Combines biuret reagent and Folin-Ciocalteau
phenol reagent which reacts with tyrosine
and tryptophan residues in proteins
Gives a bluish color which can be read at
w/length between 500 – 750 nm
More sensitive to low concentrations of
protein than biuret method
Dye Binding Methods
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A known excess of a negatively charged dye is
added to a protein solution which are positively
charged (by adjusting the pH)
Bound dye and protein form an insoluble complex
while the unbound dye remains soluble
The amount of protein present proportional to the
amount of dye that bound to it.
Turbimetric Method
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Proteins from precipitate by addition of certain
chemical e.g. trichloroacetic acid
The concentration of protein is determined by
measuring the degree of turbidity
Advantages:
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Rapid
Simple to carry out
Sensitive to low protein
concentration
Disadvantages:
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Time consuming and
laborious
Absorbance depends on
the type of protein
analyzed
difficult to quantitatively
extract proteins from
certain types of food e.g.
processed food
Other instrumental techniques
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Bulk physical properties
 Density – protein has the greatest density. So
increase in protein content will increase
density of food
 Refractive index – RI increases as protein
concentration increases
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Measurement of Adsorption of Radiation
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Infra-Red: protein absorb IR naturally due to
characteristic vibrations of certain chemical
groups
NMR: measuring the area under a peak in an
NMR chemical shift spectra that corresponds
to the protein fraction
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Measurement of Scattering of Radiation
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Light scattering: use degree of turbidity to
measure protein content
Ultrasonic scattering: ultrasonic velocity and
absorption are related to concentration of
protein aggregates present.
Advantages:
Disadvantages:
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Non-destructive
Little or no sample
preparation
Rapid and precise
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Calibration curve must be
prepared
Can only be used to
analyze foods with
relatively simple
compositions
Difficult to disentangle the
contribution that the
protein makes to the
overall measurement from
that of the other
component
Protein Separation and
Characterization
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Why need to have a knowledge of the effects
of environmental conditions on protein
structure and interaction?
1. It helps to determine the most suitable
conditions to use to isolate particular protein
from mixture of protein
2. Not select conditions which will not adversely
affect the molecular structure of the protein
A. Methods Based on Different Solubility
Characteristics
 Protein can be selectively precipitated or
solubilized by altering pH, ionic strength,
dielectric constant or temperature of a
solution
 The most simple to use when large quantities
of sample are involved
1. Salting Out
 Proteins precipitate from aqueous solutions when
the salt concentration exceeds a critical level
 Salt commonly used: Ammonium sulfate
 A two-steps procedure:
 Salt is added just below that necessary to
precipitate out interest protein.
 Salt concentration is increased to point just
above that necessary to cause precipitation
2. Isoelectric Precipitation
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Isoelectric point (pI): pH where the net
charge on the protein is zero
Proteins tend to precipitate at their pI
because there is no electrostatic repulsion
keeping them apart
Different amino acid has different pI.
3. Solvent Fractionation
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↓ in dielectric constant will ↑ magnitude of the
electrostatic interaction between charged
species
Therefore, decrease the solubility of protein in
solution
Dielectric constant can be lowered by adding
water-soluble organic solvents such as ethanol or
acetone
4. Denaturation of Contaminating Proteins
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To isolate protein that stable at high
temperature or at extremes of pH
Proteins are denatured and precipitated when
heated at high temperature or in the high
acid solution
B. Methods based on adsorption
characteristics
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Involves the separation of compounds by
selective adsorption-desorption at a solid
matrix that is contained within a column
Separation can be carried out using either
an open column or high-pressure liquid
chromatography
1. Ion Exchange Chromatography
 Relies on the reversible adsorption-desorption of
ions in solution to a charged solid matrix or polymer
network
 Positively charged is called anion – exchanger while
negatively charged is called cation – exchanger
 Two types of column are used
1. To bind protein of interest to the ion-exchange
column
2. To favor adsorption from the column
2. Affinity Chromatography
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Uses stationary phase that consists of a
ligand covalently bound to a solid support
The protein of interest binds to the ligand in
the column, and eluted using another buffer
solution
C. Methods Based on Size Differences
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Depends on the Stokes radius of a protein
Stokes radius is the average radius that a
protein has in solution
Stokes radius increases in the following
order: compact globular protein < flexible
random-coil < rod-like protein
1. Dialysis
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Use semi-permeable membranes that permit
the passage of molecules smaller than a certain
size through
A protein solution is placed in dialysis tubing
which is sealed and placed into a large volume
of water or buffer which is slowly stirred
LMW solutes flow through the bag while HMW
remains.
2. Ultrafiltration
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A protein solution is placed in a cell containing a
semi-permeable membranes and pressure is
applied to speed up the separation
LMW protein pass through the membrane
whereas the LMW molecules remains in the
solution
Use to concentrate a protein solution, remove
salt, exchange buffers or fractionate protein on
the basis of their size
3. Size Exclusion Chromatography
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Also known as gel filtration
Column which is packed with porous beads
made of a cross-linked polymeric material
Molecules larger than the pores in the beads
are excluded and move quickly through the
column whereas the movement of molecules
which enter the pores is retarded
D. Separation by Electrophoresis
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Relies on differences in the migration of
charged molecules in a solution when an
electrical field is applied across it
It can be used to separate protein on the
basis of size, shape or charge
1. Non-denaturing Electrophoresis
A buffered solution of native proteins is
poured onto a porous gel (usually
polyacrylamide, starch or agarose) and a
voltage is applied across the gel
 Gels may be contained between two parallel
plates or in cylindrical tubes
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2. Denaturing Electrophoresis
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Separate protein based on their molecular weight
Proteins are denatured prior to analysis by mixing
them with mercatoethanol and sodium dodecyl
sulfate (SDS)
Smaller protein moving more rapidly through the
matrix than larger molecules
Type of electrophoresis commonly used is sodium
dodecyl sulfate – polyacrylamide gel electrophoresis
(SDS-PAGE)
3.Isoelectric Focusing
Electrophoresis
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Protein are separated
by charge on a gel
matrix which has a
pH gradient across it
Has the highest
resolution selection of
suitable gel is
important
4. Two Dimensional Electrophoresis
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Isoelectric focusing and SDS-PAGE are used
together to improve resolution of complex
protein mixture
Proteins are separated in one direction on the
basis of charge using isoelectric focusing and
then in a perpendicular direction on the basis of
size using SDS-PAGE
E. Amino Acid Analysis
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To determine amino acid composition of
proteins
A protein sample is first hydrolyzed to release
amino acids which are then separated using
chromatography