Download 03_Physical-chemical properties of proteins

Physical-chemical properties of proteins; methods of
its determination, precipitation reactions
Separation of Amino Acids
and Proteins
1.
2.
3.
4.
5.
Chromatography – the method of separating amino
acids on the basis of differences in absorption, ionic
charges, size and solubility of molecules
Electrophoresis – effects separation in an electric
field on the basis of differences in charges carried by
amino acids and proteins under specific condition
Ultracentrifugation – effects separation on the basis
of molecular weight when large gravitational forces are
applied in the ultracentrifuge.
Precipitation Methods – salts as sodium sulfate,
ammonium sulfate, cadmium nitrate, silver nitrate and
mercuric chloride at specific conc. precipitate some
proteins while others remain in solution
Dialysis – is for the removal of small, crystalloidal
molecules from protein solution.
Chromatography
• Much of modern biochemistry depends on the use of
column chromatographic methods to separate
molecules.
• Chromatographic methods involve passing a
solution (the mobile phase) through a medium (the
immobile phase) that shows selective solute
components.
• The important methods of chromatography are:
1. Ion-Exchange Chromatography
2. Antibody Affinity Chromatography
3. Gel Filtration Chromatography
4. HPLC (High Performance Liquid Chromatography)
HPLC
Gel Filtration Chromatography
Precipitation of proteins
Proteomics
• Proteomics is the science of protein
expression of all the proteins made by a
cell
• Proteome pertain to all proteins being
made according to the transcriptome (RNA
profile). It is often visualized by a system
interaction map as seen in the
proteogram.
Procedures of the Proteomics
• Commonly used procedures by
Proteomics are:
• Mass Spectrophotometry – detects exact
mass of small peptides (molecular weight).
• X-ray Crystallography – determines 3D
shape of molecules mathematically
• NMR Spectroscopy – magnetic signal
indicates distances between atoms
Qualitative analysis of Proteins
Precipitation reactions
Colour Reactions of Proteins
Precipitation reactions
Protein exist in colloidal solution due to
hydration of polar groups (-COO, NH3+, -OH)
They can be precipitated by dehydration or
neutralization of polar groups.
Precipitation by salts
To 2 ml of protein solution add equal volume of saturated
(NH4)2SO4 solution
White precipitation is formed
Precipitation by heavy metal salts
To 2 ml of protein solution, add
few drops of Heavy Metals (lead
acetate
or
mercuric
nitrate)
solution,
results
in
white
precipitation
Precipitation by alkaloidal
reagent
To a few ml of sample solution add
1-2 ml of picric acid solution.
Formation of precipitation indicates
the presence of proteins
Precipitation by organic solvents
To a few ml of sample solution, add 1 ml of
alcohol. Mix and keep aside for 2 min.
Formation of white precipitation indicates the
presence of protein
Precipitation by heat
Take few ml of protein solution in a test tube
and heat over a flame. Cloudy white
precipitation is observed
Precipitation by acids
To 1 ml of protein solution in test tube, add few
drops of 1% acetic acid, white precipitation is
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formed
Colour Reactions of Proteins
Proteins give a number of colour reactions with different
chemical reagents due to the presence of amino acid
Biuret test
The Biuret test is a chemical test used for detecting the
presence of peptide bonds
 In
the
presence
of
peptides,
a
copper
(II)
ion
forms
violet-colored
coordination
in
an alkaline solution
To 2 ml of protein solution in a test tube add 10%
of alkaline (NaOH) solution. Mix and add 4-5 drops
of 0.5% w/v copper sulphite (CuSO4) solution
Formation of Purplish Violet Colour indicates the
presentation of proteins
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Biuret Test
Chemical Reaction in the Biuret test
Xanthoproteic Test
To 2 ml of protein solution add 1 ml conc.HNO3
Heat the solution for about 2 minutes and cool
under tap water
A yellow colour is obtained due to the nitration of
aromatic ring
Add few drops of 40% w/v NaOH solution
 The yellow colour obtained initially changes to
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orange
 Millon’s Test
When Millon’s reagent is added to a protein,
a white precipitation is formed, which turn
brick red on heating
Phenols and phenolic compounds, when
mixed with Hg(NO3)2 in nitric acid and traces
of HNO2, a red colour is produced
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Ninhydrin Test
When protein is boiled with a dilute solution
of ninhydrin, a violet colour is produced
Proteins
Hydrolysis
Amino acids
Amino Acids + Ninhydrin
Keto acid + NH3
+ CO2 + Hydrindantin
NH3 + Ninhydrin
Pink colour
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Aldehyde Test
To 1 ml of protein solution in test tube add few ml
of PDAB in H2SO4.
Mix the contents and heat if necessary.
The formation of purple colour is observed
Phenol’s reagent Test
To few ml of protein solution in a test tube add 1
ml of NaOH solution (4% w/v) and 5 drops of
phenol’s reagent.
The formation of blue coloured solution
Observed
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Color Reactions of Proteins
Test
Ninhydrin
Composition of
Reagent
Triketohydrin Hydrate
Blue or Purple
Biuret
NaOH + CuSO4
Violet
Millon’s
Hg in HNO3
Red
Xanthoproteic
Conc. HNO3
Lemon yellow
Hopkins-Cole
Glyoxylic acid and
conc. H2SO4
Conc. HCl , sucrose
Pb(OAc)2 Sullfanilic
acid in HCl + NH4OH
Violet ring
Violet
Red orange –
lighter orange
Indole group
+ Tryptophan
+ Histidine and Tyrosine
10% NaOH, ά naphtol,
alkaline hypobromite
Intense red
color
Guanidine
+ Arginine
Acree-Rosenheim HCHO conc. H2SO4
Violet ring
Indole group
+Tryptophan
Reduced Sulfur
KOH, Pb(OAc)2
Black ppt
Sulfur
Br water
Br.H2O, amyl alcohol
Pink
Indole group
+ Cystine, Cystein and
methionine
+ Tryptophan
Molisch
ά naphtol in alcoholic Violet ring
H2SO4
Glacial Acetic acid and Reddish violet
Carbohydrates
Glycoprotein
Liebermann
Erlich’s Diazo
Sakaguchi
Adamskiewez
+ Result (Color) Group Responsible
Importance
Free amino and free Test for amino acid,
COOH
peptides in determining
amino acids
Peptide linkages
+ Tripeptides up to
protein
Hydroxyphenyl
+ Tryptophan
group
Benzene ring
+ Tyrosine, Phenyl
alanine, Tryptophan
Indole group
+ Tryptophan
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Indole group
+ Tryptophan
Quantitative Analysis of Proteins
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Kjeldahl method
The Kjeldahl method was developed in 1883
by a brewer called Johann Kjeldahl
 A food is digested with a strong acid so that
it releases nitrogen which can be determined
by a suitable titration technique.
The amount of protein present is then
calculated from the nitrogen concentration of
the food
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Kjeldahl method
Principles
Digestion
Neutralization
Titration
The food sample to be analyzed is weighed into
a digestion flask
(NH4)2SO4 + 2 NaOH
2NH3 + 2H2O + Na2SO4
H3BO3 (boric acid)
NH4+ + H2BO3- (borate ion)
H+
H3BO3
Enhanced Dumas method
A sample of known mass
CO2, H2O and N2
Combustion (900 oC)
Nitrogen
Thermal conductivity detector
The nitrogen content is then measured
Methods using UV-visible spectroscopy
These methods use either the natural ability
of proteins to absorb (or scatter) light in the
UV-visible region of the electromagnetic
spectrum, or they chemically or physically
modify proteins to make them absorb (or
scatter) light in this region
Principles
Direct measurement at 280nm
Biuret Method
Lowry Method
Dye binding methods
Turbimetric method
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Direct measurement at 280nm
Tryptophan and tyrosine absorb ultraviolet
light strongly at 280 nm
The tryptophan and tyrosine content of
many proteins remains fairly constant, and so
the absorbance of protein solutions at 280nm
can be used to determine their concentration
Biuret Method
A violet-purplish color is produced when
cupric ions (Cu2+) interact with peptide
bonds under alkaline conditions
The absorbance is read at 540 nm
Lowry Method
The Lowry method combines the Biuret
reagent with another reagent (the FolinCiocalteu phenol reagent) which reacts
with tyrosine and tryptophan residues in
proteins.
 This gives a bluish color which can be read
somewhere between 500 - 750 nm depending on
the sensitivity required
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Other Instrumental Techniques
Measurement of Bulk Physical Properties
Measurement of Adsorption of Radiation
Measurement of Scattering of Radiation
Methods Based on Different Solubility Characteristics
Salting out
Isoelectric Precipitation
Solvent Fractionation
Ion Exchange Chromatography
Affinity Chromatography
Separation Due to Size Differences
Dialysis
Ultra-filtration
Size Exclusion Chromatography
Two Dimensional Electrophoresis
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Amino Acid Analysis
 Amino acid analysis is used to determine
the amino acid composition of proteins.
 A
protein
sample
is
first
hydrolyzed
(e.g. using a strong acid) to release the amino
acids,
which
are
then
separated
using
chromatography, e.g., ion exchange, affinity
or absorption chromatography.
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Name of the Test
Reagent Used
Positive results
Remarks
Biuret Test
NaOH, dilute
CuSO4
violet
+ results with
polypeptides and
proteins
Xanthroproteic
Test
Conc. H2SO4
AA with benzene
ring (yellow)
Proteins with trp, tyr,
phe
Million’s Test
Hg(NO3) and
Hg(NO2)2
Tyrosine (red)
+ phenolic compounts
Sulphur Test
Lead acetate,
dissolved with
NaOH
Gray or black
precipitate
+ lead sulphide,
formed as the result of
the decomposition of
the cysteine by the
alkali.
Hopkins Cole Test
Glyoxylic acid,
sulfuric acid
Tryptophan (violet
ring)
+ with any compound
with indole ring
Ninhydrin Test
ninhydrin
Free-NH2 group
(blue)
+ results given by NH3,
primary amines, amino
acids, peptides, and
proteins