PROTEIN ANALYSIS
Introduction 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 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
PrinciplesA. Digestion
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 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 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.
Advantages: universal high precision good
reproducibility
Disadvantages: 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 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
Advantages Faster Doesn’t need toxic
chemicals or catalysts
Easy to use Samples can be
measured automatically
Disadvantages 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 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 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 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 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 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 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: Rapid Simple to carry out Sensitive to low
protein concentration
Disadvantages: 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 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
Measurement of Adsorption of Radiation 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
Measurement of Scattering of Radiation 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: Non-destructive Little or no sample
preparation Rapid and precise
Disadvantages: 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 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 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 ↓ 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 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 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 used1. To bind protein of interest to the ion-
exchange column2. To favor adsorption from the column
2. Affinity Chromatography 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 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 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 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 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 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
2. Denaturing Electrophoresis 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
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 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 To determine amino acid composition of
proteins A protein sample is first hydrolyzed to
release amino acids which are then separated using chromatography
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