Biochemistry Experiments

Salt Precipitation and Biuret Method of Protein

Determination
 Summary
¡ Amino Acids and Peptide bonds in proteins
¡ Salt precipitation of proteins
¡ Effect of pH on proteins
¡ Absorbance protein assays
l Absorbance at 280 nm
¡ Colorimetric assays
l Biuret
l Lowry
l Bradford
l Bicinchoninic Acid
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 Protein precipitation
¡ The main purpose of protein precipitation is to separate the
protein from the solution either to eliminate interferences or to
purify them.
¡ Depending on the solubility and molecular structure of the
protein, the efficacy of various precipitation methods can be
different.
¡ When a protein is dissolved in an aqueous solution, the
surrounding water forms around proteins forming a “protein
hydration or a hydration shell
¡ The shell allows homogeneous dispersion of the protein in
solution and interactions by hydrogen bonds. This is translated
into solubility in aqueous solutions.
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 Salt Precipitation

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 Protein precipitation
¡ Certain salt are used in protein precipitation as tools to
purify proteins by precipitation.
¡ Ammonium sulfate is usually the salt of choice since it is
cheap, very soluble in water, and is able to become much
more hydrated (interacts with more water molecules) than
almost any other ionic solvent.
¡ At low salt concentrations (<0.15M) the addition of more
salt in general tends to increase the solubility of proteins as
ions shield the protein molecules from the charges of other
molecules; this trend is termed ‘salting-in’.

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 pH affects protein solubility and structure
¡ pH affects solubility and charge of proteins
¡ The change of pH will lead to the ionization of amino acids
atoms and molecules, change the shape and structure
of proteins, thus damaging the function of proteins.
¡ At a specific pH the positive and negative charges will
balance and the net charge will be zero. This pH is called
the isoelectric point, and for most proteins it occurs in the
pH range of 5.5 to 8.
¡ Without a net charge, protein-protein interactions and
precipitation are more likely.
¡ Thus, proteins have minimal solubility at their isoelectric
point
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 Salt precipitation
¡ At some point the ionic strength becomes too high and starts
to have a negative effect on the solubility of proteins,
termed ‘salting-out’.
¡ This happens because the dissolved salt competes with the
proteins for scarce water molecules, increasing the surface
tension of water and therefore causing the protein to fold
tighter.
¡ The reduction in protein surface area means less protein-
water interactions which allows for more hydrophobic
interactions between protein molecules, causing aggregation
and subsequently precipitation.

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 Criteria for choice
¡ The criteria for choice of a protein assay are
usually based on
sensitivity range
l
l Specificity
l Nature of protein being assayed
l volume/amount of sample needed
l availability of protein after assay
l presence or absence of interfering agents
l Convenience and need for accuracy

¡ Because different proteins have different amino acid

compositions, the sensitivity of colorimetric assays to
individual proteins may vary widely

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 Biuret protein assay
¡ The Biuret reaction can be used for both
qualitative and quantitative analysis of protein
¡ The Biuret reagent is made of :
¡ potassium hydroxide (KOH) and copper (II)
sulfate (CuSO4), together with potassium sodium
tartrate
¡ KOH provides an alkaline medium for the
reaction to take place

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 Biuret protein assay
Principle
¡ Molecules with two or more peptide bonds react with
Cu2+ ions in alkaline solution and form a purple
complex
¡ The nitrogen atoms of the peptide bonds form a
coordination bond with the metal ion.
¡ The purple complex absorbs maximally at 540-550nm
¡ The quantity of the complexes formed is proportional
to the number of peptide bonds

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The reaction do not occur with amino acids because the absence of
peptide bonds, and also that with di-peptide because presence of
only one peptide bond, but do with tri-, oligo-, and poly-peptides.
Biuret reaction needs presence of at least two peptide bonds in a
molecule
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The reaction takes its name "Biuret Reaction" from the fact that
biuret itself, obtained by heating urea, gives a similar colored
complex with cupric ions

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 Biuret protein assay
Analysis
¡ The determination of protein concentration is done
using a calibration curve created using samples of
known concentration
¡ Prepare a standard curve of absorbance versus
micrograms protein, and determine amounts from the
curve.
¡ Determine concentrations of original samples from the
amount protein and dilution factor, if any.

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 Biuret Protein Assay
Considerations for use
¡ The assay is reliable for protein amounts between 0.5-
20mg, consuming a lot of material
¡ The Biuret can be scaled down for smaller cuvette
sizes, consuming less protein.
¡ The principle of the biuret assay involves a single
incubation of 20 min. It can be done in a short time
¡ The advantages of the method include that only few
materials (e.g. Tris and amino acid buffers) interfere
with it
¡
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 Biuret Protein Assay
¡ The color is stable, but all readings should be taken within
10 min. of each other.
¡ The biuret is a good general protein assay for batches of
material for which yield is not a problem.
¡ It does not depend on the amino acid composition of the
protein
¡ Its disadvantages are its low sensitivity and that it requires
at least a lot of protein

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 UV absorbance assay
Principle
¡ Proteins in solution absorb ultraviolet light with
absorbance maxima at 280 and 200 nm.
¡ Amino acids with aromatic rings are the primary
reason for the absorbance peak at 280 nm.
¡ Peptide bonds are primarily responsible for the
peak at 200 nm.
¡ Secondary, tertiary, and quaternary structure all
affect absorbance, therefore factors such as pH,
ionic strength, etc. can alter the absorbance
spectrum
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 Absorbs UV light

Accounts for the characteristic strong absorbance of light

by most proteins (at 280nm)
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 Comments
¡ Cold solutions can fog up the cuvette, while
warm solutions can release bubbles and interfere
with the readings.
¡ For concentrated solutions (absorbance greater
than 1) simply dilute the solution.
¡ Absorbance coefficients of some common
protein standards:
l Bovine serum albumin (BSA): 63
l Bovine, human, or rabbit IgG: 138
l Chicken ovalbumin: 70

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 Lowry assay (Folin-Ciocalteu method)
¡ The Lowry assay (1951) can be used for protein
determination for cell fractions, chromatography
fractions, enzyme preparations e.t.c
¡ Requires many reagents including sodium
potassium tartrate, sodium carbonate, NaOH,
copper(II) sulfate
¡ Folin's reagent (a mixture of phosphotungstic
acid and phosphomolybdic acid in phenol)

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 Lowry assay
Principle
¡ Under alkaline conditions the divalent copper
ion forms a complex with peptide bonds in
which it is reduced to a monovalent ion
¡ Monovalent copper ion and the radical groups of
tyrosine, tryptophan, and cysteine react with
Folin reagent to produce an unstable product that
becomes reduced to molybdenum/tungsten blue
¡ The tungsten blue absorbs maximally at 650nm

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 Lowry assay
Analysis
¡ The Lowry method is best used with protein
concentrations of 0.01-1.0 mg/ml.
¡ Prepare a standard curve of absorbance versus
micrograms protein and determine amounts from
the curve.
¡ Determine concentrations of original samples
from the amount protein, volume/sample, and
dilution factor, if any.

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 Lowry (Folin) protein assay
¡ The advantages of the method include that it is quite
sensitive and is able to detect even 1 μg of protein.
¡ Its disadvantages are that it takes rather long to carry
out, is disturbed by various materials (including
ammonium sulphate, glycine and mercaptans)
¡ The incubation time is critical. Recording of
absorbances can be done within 10 min. of each other
¡ As different proteins contain different amounts of
tyrosine, the amount of the coloured product will also be
different.

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 Comments on Lowry method
¡ The Lowry method can be used with protein
concentrations of 0.01-1.0 mg/ml.
¡ The modification is less sensitive to interfering agents
and is more sensitive to protein than the original.
¡ It can be scaled up for larger cuvette sizes, however
more protein is consumed.
¡ Proteins with an abnormally high or low percentage of
tyrosine, tryptophan, or cysteine residues will give
high or low errors, respectively.
¡ As a consequence, this method is more suited to
compare the concentration of solutions of the same
protein than for absolute concentration measurement
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 Bradford protein assay
Principle
¡ The assay is based on the reaction of Coomassie
Brilliant Blue an acidic solution with arginine residues
and less so with histidine, lysine, tyrosine, tryptophan,
and phenylalanine residues.
¡ The absorbance shifts from 465 nm to 595 nm when
binding to protein occurs.
Analysis
¡ Prepare a standard curve of absorbance versus
micrograms protein and determine amounts from the
curve.
¡ Determine concentrations of original samples from the
amount protein and dilution factor, if any.

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 Bradford protein assay
Considerations for use
¡ Immunoglogin G (IgG - gamma globulin) is the
preferred protein standard
¡ The advantages of the method include that it is highly
sensitive
¡ It is fairly accurate and samples that are out of range
can be retested within minutes.
¡ The Bradford is recommended for general use,
especially for determining protein content of cell
fractions and assessing protein concentrations for gel
electrophoresis

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 Bradford protein assay
¡ It is able to measure 5-200 μg of protein and is very
fast.
¡ Only relatively few materials interfere with it (it
works even in presence of urea or guanidine
hydrochloride)
¡ Even traces of detergent (e.g. cleaning products) can
invalidate the results.
¡ Its disadvantages are that it depends strongly on amino
acid composition and that it stains the cuvettes used

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Bicinchoninic Acid (BCA) Protein Assay (Smith)
Principle
¡ The BCA assay is based on chemical principles similar to those
of the biuret and Lowry assays.
¡ The protein to be analyzed is reacted with Cu2+ which produces
Cu+
¡ The Cu+ is chelated by BCA, which converts the apple-green
color of the free BCA to the purple color of the copper- BCA
complex.
¡ Samples of unknown protein and relative standard are treated
with the reagent, the absorbance is read in a spectrophotometer at
562 nm
¡ Concentrations are determined from a standard calibration curve

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 BCA
Considerations for use
¡ This assay has the same sensitivity level as the Lowry and
Bradford assays.
¡ Its main advantages are its simplicity and its usefulness in
the presence of 1% detergents such as Triton or sodium
dodecyl sulfate (SDS).
¡ The BCA reagent is fairly stable under alkaline conditions

¡ The bicinchoninic acid (BCA) assay is available in kit

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