PROTEIN Name of group

1. M. Badrul Uyun
The purpose of experiment
1) Distinguishing the solubility
nature of proteins in reversible and
16030194023
irreversible.
2) Distinguishing protein
2. Hanifatin Azizah
denaturation reactions caused by 16030194086
acids, salts and salts from heavy
metals, as well as heating based on
observation.
3. Putri Anggraeni R
3) Understanding the causes of 16030194089
precipitation in protein.
4) Identifying the presence of
proteins through a color reaction.
Preparing tools and materials

Tools Materials
Preparing protein solution from quail egg

White egg
Broken eggs Doing dilution (2 ml
solution
white egg + aquades
until 10 mL)
1. Denaturation of protein
The objective of experiment is test several factors that cause denaturation of proteins.
a. Denaturation because of adding CH3COOH

5 ml protein solution Added 2 drop White egg solution after adding 2 drops
CH3COOH of CH3COOH become white solution
and turbid
 Solution become
Milk after adding 2 drops of CH3COOH Heated until 5
white turbid solution
become white solution and turbid minutes
and flake form
the reaction is:
Conclusion :
Protein will denatiration with adding acid
that is proved flake form in the solution .
 1. Denaturation of protein
b. Denaturation because heating

3 ml of protein Heating until 1 Divided into 2 part part A,

minutes , still white Heated again
solution added 3 drops of
solution (NH4)2SO4 part B without
adding (NH4)2SO4 , white
turbid solution
 The reaction is:

Tube 1  milk +
Tube 1  white egg
(NH4)2SO4 : white
solution + (NH4)2SO4 :
Conclusion :
turbid solution Protein will denaturation with increasing
white turbid solution
+Heated : white turbid temperature that proved by precipitate
+Heated : white turbid
solution (+), flake form
solution (+), flake form formed in the solution.
Tube 2  Heated :
Tube 2  Heated :
turbid solution and
unchanging
white precipitate
 1. Denaturation of protein
a. Denaturation because of adding formaldehyde

• Mixture solution + milk:

1,5 ml formaldehyde +
Added drop by drop of
2 ml aquades, colorless
white egg solution
solution
Added drop by drop of white turbid solution,
milk white precipitate
• Mixture solution + white
egg: turbid solution and
little precipitate
The reaction is:

Conclusion :
Protein will denaturation with adding
formaldehyde that is proved by
precipitate formed in the solution.
 2.TheAmfoter Characteristic of protein
objective of experiment to determine the amphoteric properties of proteins (can
be both alkaline and acidic), by reacting proteins with acidic solutions and alkaline
solutions.
a. Test in acid condition

3 ml of aquadest + HCl + Congo Added 2 ml of protein (egg) = Added 2 ml of protein (milk) =

indicator = purple solution red purple solution red purple solution
Amfoter Characteristic of protein
• Test in base condition

3 ml of NaOH + 2 ml of protein (egg) + 2 ml of protein (milk)

phenolptalien = pink NaOH + phenolptalein + NaOH +
solution = pink solution phenolptalein= pink
solution
Reaction
• In acid condition
Conclusion :
Protein can react with acid, that is proved
with color changing of congo indicator

• In base condition Conclusion :

Protein can react with base, that is proved
with color changing of PP indicator.
Protein is amphoteric compound, because
can react with acid or base.
3. Precipitation of protein
The purpose of the experiment is to understand the cause of precipitation
process on protein.
a. Precipitation of protein with ammonium sulphate
Egg solution

4 ml of Milk solution entered Added 4 ml ofammonium Added 3 ml of aquades =

into test tube sulphate = turbid solution, precipitate dissolve
white precipitate
Milk solution

4 ml of Egg solution Added 4 ml of ammonium Added 2 ml of aquadest =

entered into test tube sulphate = white turbid solution, precipitate dissolve
white precipitate
 Reaction
H2O
H2O
+
O
H3N CH C
R2
-
(NH4)2SO4, Kalor
+
O
R2 H2O
+
O
R2
Conclusion :
N C COO H3N CH C H3N CH C
R H (aq) -H2O R
N C COO- N C COO- Precipitation of
H H (s) R H (aq)
H H protein with adding
H 2O

H 2O
ammonium sulphate
hidration of protein
hidration of protein hidration of protein
is reversible. It is
proved with
precipitate dissolve
when adding
aquades.
3. Precipitation of protein
• Protein precipitation with mineral acid
- reacted with HCl

2ml of concetrated HCl entered into test Added 1,5ml of milk solution and egg
tubes solution
After adding egg solution, let it stand Added HCl again = precipitate dissolve
until form white ring
 Reaction
Conclusion :
O R2 O R2 Precipitation of
H protein with adding
-+ H -+
Cl NH3 C N C COOH + HCl(aq) Cl NH3 C N C COOH HCl is reversible. It is
H (s) H (aq)
proved with
precipitate dissolve
when adding
aquades.
 Precipitation protein
a. Precipitation of protein with mineral acid
- reacted with HNO3

1 ml of HNO3 solution entered into test Added HNO3 again until form white
tube Added milk solution formd white ring precipitate
HNO3 entered into test tube
 Reaction

Conclusion :
Protein with HNO3 is irreversible. It is proved that
precipitate increase when added HNO3 again.

.
3. Precipitation protein
• Protein precipitaton with heavy metals

1,5 ml protein + Added CuSO4 1,5 ml protein +

CuSO4 (blue precipitation dilute ZnSO4 (blue
precipitation) precipitation)
Added ZnSO4 1,5 ml protein + Added PbSO4 1,5 ml protein + Added FeSO4
precipitation PbSO4 (blue precipitation FeSO4 (blue precipitation
dilute precipitation) dilute precipitation) dilute
 Conclusion :
Precipitation of proteins with heavy metals is
reversible. Characterized by the formation of the
precipitate then can be dissolved again.

1,5 ml protein + HgSO4 Added HgSO4 .

(blue precipitation) precipitate dilute
 4. Colour Reaction
a.Biuret reaction
The biuret test is a chemical test used for detecting the presence
of peptide bonds

• Milk + NaOH 40% : • Milk + 0,5% CuSO4 :

3 ml sample protein white solution purple solution ++
Added 1 ml NaOH 40% • White egg sol + • White egg sol. +
solution
NaOH 40% : still 0,5% CuSO4 : purple
colorless solution +++
The reaction is:
CuSO4 (aq) + 2NaOH (aq) Cu(OH)2 (aq) + Na2SO4(aq)
Cu(OH)2 Cu2+ + 2OH- O
H H2
N C H C
O
C N C
H2
H H2
N C C + Cu2+ O
2 Cu2+
C N C
H2 H O
H2
O N C C
H C N C
H2
H
O

• Conclusion :
Protein solutions form peptide bonds are characterized by
positive test results of the formation of purple solution on
the biuret test because complex compounds have been
formed between Cu2+ and N of the peptide bond molecule.
 4. Colour Reaction
b. Xanthoprotein reaction

Sample protein After sample protein Added NH3

solution + HNO3 solution + HNO3 After Heated : yellow
concentarte concentarte, become solution
white turbid solution
 The reactions is:
O
O

H 2N C
H 2N C
CH OH
CH OH

O 2N NO2 O 2N NO2
+ 3H N O 3 + 3H 2O

NO2 NO2

After added NH3,

become orange solution
• Conclusion :
The protein solution contains a
benzene ring characterized by the
formation of an orange solution in the
xanthoprotein reaction test .
• 4. Colour reaction
Nynhidrin reaction
the objective of the experiment is to detecting the existence of protein, peptides
and amino acids.

Egg solution and milk solution entered into Added ninhydrin solution. the result is
test tube colorless solution
Hetaed on 100℃ for 10 minutes Egg solution + ninhydrin + heated : Rhuheman’s purple
Milk solution+ ninhydrin + heated : Rhuheman’s purple
Reaction Conclusion :
The protein solution contains free
amino acids characterized by the
formation of a purple solution on the
Ninhydrin reaction test.
 4. Colour Reaction
d. Millon reaction
to identify amino acids in proteins containing phenol groups, such as tyrosine

3 ml sample protein Added millon Millon reaction (sample Millon reaction (sample + millon
solution reagent, become + millon reagent + reagent + heated + NaNO3 1% :
white solution heated) : white pink solution Heated again) : red
precipitate formed and brick precipitate and red brick
a little pink solution solution.
The reaction is:

• Conclusion :
The protein solution contains tyrosine,
characterized by the formation of red
brick precipitate.
 4. Colour Reaction
e. Hopkin-Cole reaction
The objective of experiment is detecting the presence of
tryptophan in proteins

1 ml sample protein solution + +H2SO4 : 2 layer

added HgSO4 reagent: formed
1 drop formaldehyde : white
white solution
solution for milk and still
colorless for white egg solution
The reaction is :

• Conclusion :
In a protein solution do not contain the indol nucleus
of tritofan with aldehyde characterized by the
absence of purple rings
5. Hydrolysis of protein and sulphur identification
This experiment aims to prove the presence of sulfur in proteins in a solution
of milk.

1 ml protein solution + 1 Heated in the water After heating : yellow After adding Pb-asetic :
ml NaOH 40% : white bath solution blackish brown (+) for
solution for milk and milk and blackish
colerless for white egg brown (++) for white
solution egg solution
The reactions is:
Pb2+ + 4 OH-  PbO22- + 2 H2O
S2- + 2 H2O + PbO22-  PbS↓ + 4 OH-
Black precipitate

Conclusion :
protein solution containing sulfur is
characterized by the formation of black
precipitate in the solution