Protein Qualitative Tests and Precipitation Methods
Qualitative tests of protein
Certain functional groups in proteins can react to produce characteristically colored products.
-The
color intensity
of the product formed by a particular group varies among proteins in
proportion
to the number of reacting functional or free groups present and their accessibility to the
reagent.
Biuret test
:
This test is specific for the
peptide bond
. Substances containing not less than two peptide linkages give
this test.
Objective:
detect the presence of peptides or proteins in a sample.
Principle:
When proteins and peptides (i.e peptide bonds) treated with an
alkaline
solution of
dilute copper
sulfate
a violet color
is formed . A positive test is indicated by the formation of a
violet color
.
Note:
the color density is
proportional
to amount of proteins present.
*
biuret reagent
is
alkaline copper sulfate solution
.
Method:
1- Add 2ml of protein Albumin
2- Add 1 ml of biuret reagent and mix well.
Result:
Protein precipitation
•
Is widely used in downstream processing of biological products in order
to concentrate
proteins
and
purify
them from various contaminants.
•
The
solubility of proteins
is affected by pH, temperature, salts, heavy metal salts etc.
1.Effect of salt concentration on the protein solubility:
Objective:
to investigate the effect of different salt concentration on protein solubility.
Principle:
The
low salt concentration
solutions make protein
solubility easier
using the attraction of salt ions to the
functional groups of the protein (
salting in
).
On contrast,
high salt concentration
or solids dissolved in the reaction medium up till saturation solutions
causes the protein to
precipitate s
ince salt ions, in this case, compete with the protein molecules in binding
water molecules (
salting out
).
So the salt it just cause
protein dehydration
.
e.g (NH4)SO4
Note:
Each protein can be precipitated at specific salt concentration.
There is
inverse relationship
between the Mw of protein and the concentration of salt)
High
Mw need
low
concentration salt ( low percentage of saturation)
Low
Mw need
high
conc. of salt ( High percentage of saturation)
Egg Proteins:
Albumin
and
globulin
Separated by centrifugation at 3000 for 20 min.
The
albumin
is the
supernatant
because it is has
low Mw
. and
globulin
is the
precipitate which is
higher Mw
than albumin.
+ NaCl
(low salt concentration
Globulin dissolves (salting in)
+
50% saturated
(NH
4
)2SO
4
Globulin precipitate (salting out)
Globulin has high MW so it precipitate at lower
salt concentration
+
50% saturated
(NH
4
)2SO
4
No precipitation
Albumin has low MW so it need higher salt
concentration to precipitate
+
100% saturated
(NH
4
)2SO
4
Albumin precipitate (salting out)
Albumin has low MW so it precipitate at high
salt concentration
Method:
Result:
2.Precipitation of proteins by acids:
Objective:
To investigate the effects of strong acids on the protein solubility.
Principle:
This test depend on affecting solubility of the protein as a function of
changes in pH
. In highly
acidic
media
, the protein will be
positively
charged, which is attracted to the acid
anions
that cause them to
precipitate.
Method
Result:
Precipitation of albumin using
Trichloroacetic acid [TCA]
Precipitation of albumin using
concentrated nitric acid.
3. Precipitation of protein by salts of heavy metals:
Heavy metal salts
usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic
weights. Since salts are ionic they
disrupt salt bridges
in proteins. The reaction of a heavy metal salt
with a protein usually leads to an insoluble metal protein salt.
Objective:
to identify the effect of heavy metal salt on protein.
Principle:
-Heavy metal salt will
neutralize
the protein:
By the
negative
charge of protein will bind with
positive
charge of metal ion . Then the protein will
precipitate as insoluble metal protein salt .
Method:
Result:
4. Protein denaturation:
Objective:
to investigate the effect of high temperature on protein structure.
Principle:
Non-covalent bond can be broken by heating, leading to protein denaturation and the precipitation.
Denaturation
is a process in which the proteins losing its quaternary structure, tertiary structure and
secondary structure, by application of some external factor or compound such as a strong acid or base, a
conc. inorganic salt, an organic solvent (e.g., alcohol or chloroform), or heat.
- without alteration of the molecule's primary structure, i.e., without cleavage of any of the primary chemical bonds that link one
amino acid to another.
Method:
1- Take 1 ml of protein Albumin .
2- Place it in a boiling water bath for 5-10 minutes.
3-Remove aside to cool to room temperature.
4-Note the change.
Result:
Questions :
Do you think free amino acids will give a positive result with biuret test ? why?
What is the least number of amino acids bonded together by peptide bonds that will respond positively to
biuret test?
After heating albumin at high temperature, does it still biologically active? Why?
Can we use salting out method in fractionating mixture of proteins? Explain your result with example.
Qualitative tests of proteins involve reactions producing colored products, such as the Biuret test specifically detecting peptide bonds. Protein precipitation methods are used for concentration and purification, affected by factors like pH, temperature, salts, and heavy metals. Different techniques like salt concentration effects and protein denaturation play roles in these processes.
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Presentation Transcript
Certain functional groups in proteins can react to produce characteristically colored products. -The color intensity color intensity of the product formed by a particular group varies among proteins in proportion proportion to the number of reacting functional or free groups present and their accessibility to the reagent.
Biuret test Biuret test: This test is specific for the peptide bond this test. Objective: Objective: detect the presence of peptides or proteins in a sample. peptide bond. Substances containing not less than two peptide linkages give Principle: Principle: When proteins and peptides (i.e peptide bonds) treated with an alkaline sulfate a violet color is formed . A positive test is indicated by the formation of a violet color alkaline solution of dilute copper violet color. Note: Note: the color density is proportional proportional to amount of proteins present. * biuret reagent biuret reagent is alkaline copper sulfate solution.
Method: Method: 1- Add 2ml of protein Albumin 2- Add 1 ml of biuret reagent and mix well. Result: Result: https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcT1sz0mN0h9Y4Fpri5ndOSG6mYc9fM3draaEKkxQmrfrpY3Vqxu
Protein precipitation Protein precipitation Is widely used in downstream processing of biological products in order to concentrate proteins and purify them from various contaminants. The solubility of proteins solubility of proteins is affected by pH, temperature, salts, heavy metal salts etc. Which sample would you rather inject?
1.Effect of salt concentration on the protein solubility 2.Precipitation of proteins by acids. 3. Precipitation of protein by salts of heavy metals. 4. Protein denaturation.
1 1.Effect of salt concentration on the protein solubility: .Effect of salt concentration on the protein solubility: Objective: Objective: to investigate the effect of different salt concentration on protein solubility. Principle: Principle: The low salt concentration low salt concentration solutions make protein solubility easier functional groups of the protein (salting in On contrast, high salt concentration high salt concentration or solids dissolved in the reaction medium up till saturation solutions causes the protein to precipitate s precipitate since salt ions, in this case, compete with the protein molecules in binding water molecules (salting out salting out). solubility easier using the attraction of salt ions to the salting in). So the salt it just cause protein dehydration Note: Note: Each protein can be precipitated at specific salt concentration. protein dehydration. e.g (NH4)SO4 There is inverse relationship inverse relationship between the Mw of protein and the concentration of salt) High High Mw need low low concentration salt ( low percentage of saturation) Low Low Mw need high high conc. of salt ( High percentage of saturation)
Egg Proteins: Egg Proteins: Albumin Albumin and globulin Separated by centrifugation at 3000 for 20 min. The albumin albumin is the supernatant supernatant because it is has low Mw precipitate which is higher Mw higher Mw than albumin. globulin low Mw. and globulin globulin is the
+ 50% saturated (NH4)2SO4 No precipitation + NaCl (low salt concentration Globulin dissolves (salting in) Albumin has low MW so it need higher salt concentration to precipitate + 50% saturated (NH4)2SO4 + 100% saturated (NH4)2SO4 Globulin precipitate (salting out) Globulin has high MW so it precipitate at lower salt concentration Albumin precipitate (salting out) Albumin has low MW so it precipitate at high salt concentration
Method: Method: T1 T2 T3 Take your globulin sample Take 2 ml of your albumin sample Take your T2 tube Add 4 ml of NaCl solution to your globulin tube Slightly add 1 ml of 50% saturated (NH4)2SO4 solution Add a few amount of 100% solid (NH4)2SO4 Shake it well and write your observation record your observation . Shake it well and write your observation Result: Result: Tube Observation Comment Globulin + NaCl Albumin+50% saturated (NH4)2SO4 (Albumin+50% saturated (NH4)2SO4 ) + 100% saturated (NH4)2SO4
2 2.Precipitation of proteins by acids: .Precipitation of proteins by acids: Objective: Objective: To investigate the effects of strong acids on the protein solubility. Principle: Principle: This test depend on affecting solubility of the protein as a function of changes in pH. In highly acidic media media, the protein will be positively charged, which is attracted to the acid anions that cause them to precipitate. acidic
Method Method A B In a test tube, put 3ml of conc. nitric acid carefully Put 3 ml of the albumin solution Precipitation of albumin using Trichloroacetic acid [TCA] Using a dropper add to (albumin) on the inner wall of the tube to form a layer up the acid add 5-7 drops of T.C.A solution carefully Record your observation Record your observation Result: Result: Tube Observation Comment Precipitation of albumin using concentrated nitric acid. Conc. HNO3 + Albumin Albumin + TCA
3 3. Precipitation of protein by salts of heavy metals: . Precipitation of protein by salts of heavy metals: Heavy metal salts Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt. Objective: Objective: to identify the effect of heavy metal salt on protein. Principle: Principle: -Heavy metal salt will neutralize By the negative charge of protein will bind with positive charge of metal ion . Then the protein will precipitate as insoluble metal protein salt . neutralize the protein:
Method: Method: Result: Result:
4 4. Protein denaturation: . Protein denaturation: Objective: Objective: to investigate the effect of high temperature on protein structure. Principle: Principle: Non-covalent bond can be broken by heating, leading to protein denaturation and the precipitation. Denaturation Denaturation is a process in which the proteins losing its quaternary structure, tertiary structure and secondary structure, by application of some external factor or compound such as a strong acid or base, a conc. inorganic salt, an organic solvent (e.g., alcohol or chloroform), or heat. - without alteration of the molecule's primary structure, i.e., without cleavage of any of the primary chemical bonds that link one amino acid to another.
Method: Method: 1- Take 1 ml of protein Albumin . 2- Place it in a boiling water bath for 5-10 minutes. 3-Remove aside to cool to room temperature. 4-Note the change. Result: Result:
Questions : Questions : Do you think free amino acids will give a positive result with biuret test ? why? What is the least number of amino acids bonded together by peptide bonds that will respond positively to biuret test? After heating albumin at high temperature, does it still biologically active? Why? Can we use salting out method in fractionating mixture of proteins? Explain your result with example.
