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BIOCHEMISTRY LAB
TESTS FOR PROTEINS

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BiochemistryLab/Proteins
Amino acids are among the best known components of living organisms.
They are derived from organic acids, There are more than 300 different amino
acids described . only 20 commonly occur in almost all proteins. The presence
and location of amino acids in the structure of protein molecules is genetically
determined. A fragment of the amino acid molecule, composed of the carbon,
the α-amino group and the α-carboxyl group is a common structural element of
all protein amino acids . At physiological pH (about 7.4), most of the carboxyl
groups are dissociated, create anion -COO- , and most of the amino groups
bind H+ creating cation -NH3+. Under these conditions, the dominant form of
the amino acid is therefore a zwitterion, which has two opposite electric
charges.
Most of the tests are for the indication of R in the amino acids because most
of the amino acids have carboxylic group , amine group and hydrogen . but
different R groups . Protein tests are :
1-Ninhydrine test : General test for protein .
Indication / Purple or violet complex .
2- Elements of protein : to determine the protein composed of what .
Indication / depends on reagent used .
3- Biuret test : to indicate peptide bond .
Indication / cupper coordination complex ( violet color ) .
4- Xanthoproteic test : to indicate the aromatic amino acids .
Indication /
5- Rosenheim test : test for indole ring .
Indication / Brown color ring .
6- Denaturation of protein : involves the destruction of its spatial
structures while retaining the primary structure
indication / formation of precipitate depends on the reagent used .

3.
BiochemistryLab/Proteins
1-Ninhydrine test : general test for proteins .
- ninhydrine consists of 585 amino acids that bonded together by peptide
bond .- this test is common in all amino acids because its acts in the basis of
protein not amino acids .
t reacts with amino acids of theninhydrine is the strong oxidizing agent tha-
.purple complexprotein and produces
1-using two test tubes , the first one we adding albumin and the second one
adding fructose .
2-we add 5 drops of ninhydrine for both test tubes.
3-put them into the water bath for 5 mins
4- determine wether the purple complex is formed or not .
2- Elements of protein : this test is applied to make sure that the amino
acid of the protein contain carbon atom , amino group , carboxylic group and
also to indicate sulfur .
-To determine the carbon , put the sample in test tube and heat it on the hot
plate , the Black color is observed ( black burned color ) which it means this
sample contain carbon atom .
- To determine the oxygen or carboxylic group in the sample , put your sample
in the test tube and heat it on the hot plate , the bubbles or vapor is formed
inside the test tube which it means the sample contains oxygen or carboxylic
group .

4.
BiochemistryLab/Proteins
- To determine the amino group , add a litmus paper into the sample in test
tube ( red litmus paper is changed to blue or blue litmus paper remains blue )
means that the sample contain Nitrogen or amino group .
- To determine the Sulfur , put filter paper into lead acetate then into the
sample (albumin) , the black ppt is formed (PbS) which it means the side
chain of the amino acid contains sulfur .
Note / Many amino acid side chains contain sulfur , that’s why we do this
procedure .
3-Biuret Test : this test is applied to indicate peptide bond within protein .
Amino acids are building blocks of the proteins that are linked together by
peptide bonds .
-Reagents in this test are CuSo4 and NaOH.
-CuSo4 ( Chelating agent ) is the source of CU++
-NaOH is to raise the Ph of the medium and reach the solution to alkaline
level . the aim of this alkaline solution is for the reaction to occurs.
, Cu++ions will formwhen peptide bonds are present in the alkaline solution-
from peptide bond.trogen atomscoordination complex with four ni
-As the number of peptide bond increase , the more intense the change .
-This test gives positive result when 2 or more than 2 peptide bond available.
If you do this test for a single amino acid , it will give you negative result .
Procedure :
Add 30 drops oof albumin into test tube .
Then add 5 drops of CusO4
And then 10 drops of NaOH .

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BiochemistryLab/Proteins
4- Xanthoproteic test : Test for aromatic amino acid .
Aromatic amino acids are L-tyrosin , Tryptophan and Phenyl Alanine .
-Reagents used are HNO3 and NaOH .
HNO3 is used to for nitration to react with the ring .-
-If the reaction occurred the xanthoproteic acid or nitrogen derivate is formed
which it has yellow color.
-Then NaOH is used to produce a salt ( Orange color salt ).
Procedure :
Add 30 drops of albumin into test tube .
Add 5 drops of nitric acid then heat and shake ( in order for the solution not to
coagulate.)
Finally add 20 drops of NaOH.
5- Rosenheim Test : Test for indole ring .
-Specific test for tryptophan , because tryptophan is the only amino acid that
contain indole test .
Reagents used are F3Cl3 , CH2O ( formaldehyde ) and H2SO4-
FeCl3 gives the color to the solution-
-CH2O breaks the bond to differentiate amino acids .

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BiochemistryLab/Proteins
Until H2SO4 is added , the solution doesn’t show any brown ring .-
-H2SO4 causes the formation of two layer in the solution , also brown ring is
seen between these two ring .
Above layer is protein and the lower layer is H2SO4 .-
-Note that this brown ring is not the indole ring , its just a product of the
reaction which means the amino acid contain indole ring.
Procedure :
Add 5 drops of CH2O and 3 drops of FeCl3
Mix the these two reagents together which will form rosenheim reagent.
Then add 30 drops of albumin and then shake . until here we doesn’t have
any product or result .
Add 20 drops of H2SO4 drop by drop ( DON’T SHAKE ) then observe the
formation of the colored ring ( brown ring ).
6- Denaturation of Protein : involves the destruction of its spatial
structures ( secondary & tertiary structure of protein ) while retaining the
primary structure because this reaction is not strong enough to break the
peptide bond between amino acids .
This reaction doesn’t break peptide bonds , it just breaks the hydrogen and
covalent bonds .

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BiochemistryLab/Proteins
Denaturation of protein is applied by :
1-Heating : 15 drops of albumin is added to a test tube then put the test tube
on the hot plate , the result will be a white ppt. the heating causes to destroy
the structure of protein this is not about charge .
2-Strong Acid : 15 drops of albumin is added to test tube and then HNO3 or
H2SO4 is added , then the white ppt. is formed
If HNO3 is used , the test called Heller's Test .
3- Alkaloidal reagent : such as Picric acid , the alkaloidal reagent has a
negative charge that will react with the positive part of the amino acid ( NH3+ )
And neutralize the protein , reaches the isoelectric point in which the net
charge equals to zero .
Finally yellow ppt. will form.
4- Heavy metals : such as lead acetate , ferric chloride , cupper sulfate .
Heavy metals are positively charged and this will react negative part of amino
acid ( COO- ) and neutralize the protein , reaches isoelectric point .
Lead Acetate ( Pb(Ch3coo) ) will form white ppt.
Ferric chloride ( Fecl3 ) will form yellow to brownish ppt.
Cupper sulfate ( Cuso4 ) will form blue ppts .
and second one containtyrosinif you have two samples , one contain-1
how you can differentiate ?tryptophan
2-Formaldehyde is used in which test and why ?
3- how you can indicate aromatic amino acids from other amino acids ?
November9, 2017 , Thursday Bryar R. Aliruss

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BiochemistryLab/Proteins