I.

Experiment Title : Learn the characteristic and colour reaction from

Protein
II. Experiment (start – finish) : Tuesday, April 3 2018 (10.20 am – 3.30 pm)
III. Experiment Purpose :
1) To differentiate the characteristic solubility of protein’s reversibly and
irreversibly
2) To differentiate the characteristic solubility of protein’s reversibly and
irreversibly
3) To differentiate denaturation reaction of protein its caused by acid, salt and salt
from heavy metals, and heating based on observation.
4) To understand causes precipitation in protein with colour reaction
IV. Basic Theory
Definition of protein
Protein are essential compound of animal organism. Regardless of their function,
all proteins have fundamentally similar srtucture and are made up of many amino acids
linked together in a long chain. When protein hydrolized formed amino acid.

Reaction 4.1 Hydrolysis of protein (Fessenden, 1982)

The general structure from amino acid is shown in Figure below. Note that all
amino acids contain carboxylic acid groups (-COOH), amino groups (-NH2), and
substituent, or replaceable, side chains (-R) (Milio, Frank R, 1995)
 Picture 4.1 The structure of Amino Acid (Milio, Frank R, 1995)

For classified, chains with fewer than 50 amino acids are often called peptides,
while the term protein is generally used for longer chains. (Mc Murry, John E, 2012).
Amino acids incorporated into proteins are covalently linked by peptide bonds. (Milio,
Frank R, no year)

Proteins that provide all the essential amino acids in about the right proportions
for human nutrition are called complete proteins. Examples of complete proteins are
those in meat, fish, milk, and eggs. About 50 g of complete protein per day is adequate
for adult humans. Proteins that are severely deficient in one or more of the essential
amino acids are called incomplete proteins. If the protein in a person’s diet comes
mostly from one incomplete source, the amount of human protein that can be synthesized
is limited by the amounts of the deficient amino acids. Plant proteins are generally
incomplete. Rice, corn, and wheat are all deficient in lysine. Rice also lacks threonine,
and corn also lacks tryptophan. Beans, peas, and other legumes have the most complete
proteins among the common plants, but they are deficient in methionine. (Wade, L.G,
2013)

The primary structure of a protein is the sequence of amino acids in the chain and
the location of all the disulfide bridges. Secondary structures are regular conformations
assumed by segments of the protein’s backbone when it folds. The tertiary structure is
three dimensional structure of the entire polypeptide. If a protein has more than one
polypeptide chain, it also has a quarternay structure. The two main categories of protein
tertiary structure are fibrous and globular. Fibrous proteins are bundles of elongated
filaments of protein chains and are insoluble in water. Globular proteins are
approximately spherical and are either soluble or form colloidal dispersions in water.
(Carey, 2008) The quarternary structure is the way the individual polypeptide chains are
arranged with respect to one another. The way a protein chain is folded, its tertiary
structure, affects both its physical properties and its biological function. (Bruice, Paula
Yurkanis, 2011)
 Figure 4.2 The Structure of Primary, Secondary, Tertiary, and Quarteary of Protein
(Wade,2013)

Dipolar Ion Formation

Amino acid contains a basic amino group and an acidic carboxylic group in the
same molecule. An amino acid undergoes an internal acid-base reaction to yield a dipolar
ion, also called a zwitterion (from German zwitter, “hybrid”). It caused amino acid have
unusual properties, there are : (Wade, 2013)

1) Amino acids have high melting points, generally over 200 °C

2) Amino acids are more soluble in water than they are in ether, dichloromethane, and
other common organic solvents.
3) Amino acids have much larger dipole moments than simple amines or simple acids.

4) Amino acids are less acidic than most carboxylic acidsandless basic than most
amines. In fact, the acidic part of the amino acid molecule is the group, not a group.
The basic part is the group, and not a free group
Amphoterism of Amino Acids
Fessendens, 1987 An amino acid contains both a carboxylate ion (-CO2-) and an
ammonium ion (NH3+) in the same molecule. Therefore, an amino acid is amphoteric; it
can undergo reaction with either an acid or a base to yield a cation or an anion,
respectively.
In acid :

Reaction 4.2 reaction protein with acid (Fessendens 1987)

In base :

Reaction 4.3 reaction protein with acid (Fessendens 1987)

The solubility of amino acids and proteins is largely dependent on the solution
pH. The structural changes in an amino acid or protein that take place at different pH
values after the relative solubility of the molecule. In acidic solutions, both amino and
carboxylic groups are protonated. Figure below shows an amino acid in acidic, neutral,
and basic solutions. (Millio, Frank R, 1995)
 Picture 4.3 Proteins in acidic; neutral; basic condition (Millio, Frank R, 1995)

Reaction the colour of Protein

1. Biuret Test
The biuret test used to identify the presence of protein in solution with a deep
violet color. In alkaline condition, biuret reacts with compounds containing two or
more peptide bonds to form complexes of violet colour.(Himedial Laboratories, No
year). Two peptide bonds are at least required for the formation of this complex , this
is why amino acids give negative results with Biuret test.

Reaction 4.4 Biuret Test (Joshy dan Saraswat 2002)

2. Xanthoprotein Test

The Xanthoproteic test utilizes a nitration reaction which identifies the

presence of activated benzene ring. The end product of this reaction is yellow and for
that the name comes as in Greek ‘Xanthos’ means yellow. Aromatic groups of either
the free amino acid or protein undergo nitration on heating with concentrated nitric
acid (HNO3) and forms yellow coloured product. The salts of these derivatives are
orange in colour. (Himedia Laboratories, no year). The amino acids tyrosine and
tryptophan contain activated benzene rings and readily undergo nitration. The
 phenyalanine also contains a benzene ring, but the ring is not activated and therefore
does not readily undergo nitration. The yellow solution as the result of xanthoprptein
deepens when the reaction occurs in basic solution.(Millio, Frank R, 1995)

Reaction 4.5 Xanthoprotein Test (Milio Frank R,1995)

3. Ninhydrin Test

Ninhydrin is a common reagent for visualizing spots or bands of amino acids

that have been separated by chromatography or electrophoresis. When ninhydrin
reacts with an amino acid, one of the products is a deep violet, resonance-stabilized
anion called Ruhemann’s purple. Ninhydrin produces this same purple dye regardless
of the structure of the original amino acid. (Wade, 2013). The side chain of the amino
acid is lost as an aldehyde. Treatment of the paper with ninhydrin and pyridine
causes these secretions to turn purple, forming a visible fingerprint. The mechanism
are,
 Reaction 4.6 Nynhyrin Test (Bruice, Paula Yurkanis, 2011)

The reaction of amino acids with ninhydrin can detect amino acids on a wide
variety of substrates. For example, if a kidnapper touches a ransom note with his
fingers, the dermal ridges on his fingers leave traces of amino acids from skin
secretions.

4. Millon Test

Millon’s Test is a test specific for tyrosine, the only amino acid containing a
phenol group, a hydroxyl group attached to a benzene ring. In Millon’s test, the
phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the
nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form
either a red precipitate or a red solution, both positive results. Proteins that contain
tyrosine will therefore yield a positive result. However, some proteins containing
tyrosine initially form a white precipitate that turns red when heated, while others
form a red solution immediately. Both results are considered positive. Note that any
compound with a phenol group will yield a positive test, so you should be certain that
the sample that you are testing does not contain any phenols other than those present
in tyrosine. (Milio, Frank R,1995)

Tyrosine is one of the 20 essential amino acid needed by human.(Carey,

2008). Tyrosine found in chicken, turkey, fish, milk, yogurt, cottage cheese, cheese,
 peanuts, almonds, pumpkin seeds, sesame seeds, soy products, lima beans, avocados,
and bananas.

Reaction 4.7 Millon’s Test

5. Hopkine – Cole Reaction

Hopkine-Cole test is specific for indole of aminotriptofan acid, indicated by

the formed of purple ring on a sample. (Poedjiadi, 2007). The hopkine – cole reagent
only react with proteins containing tryptophan. The protein solution is hydrolyzed by
the concentrated sulfuric acid at the solution interface. Once the tryptophan is free, it
reacts with the glyoxylic acid to form the violet product (Milio, Rank R, 1995).
Tryptophan is one of the 20 essential amino acid that needed by
human.(Carey,2008). Meanwhile the functional group of tryptophan, indole only
belongs on this amino acid. Tryptophan can be found on yolk of egg.
 Picture 4. 4 Structure of Tryptophan (Carey, 2008)

Reaction 4. 8 Hopkine – Cole Test

Protein Precipitation

Functional groups of protein (NH2, NH, OH, CO) and double ion form (zwitter
ion) bonding with water molecule with hydrogen bonding. Precipitation happened when
the protein added with chmeical substance, such as salts, organic solvent that can changes
the solubility properties of protein.

a. Precipitation of Amonium Sulphate

Ammonium sulphate is one of the reagent that caused reversible denaturation of

protein. This denaturation indicated by the formation of white precipitate. Principle
of protein precipitation with ammonium sulphate is decrease the solubility of
proteins in to water because of water adsorbed by salt. Ammonium sulphate taht
diluted into water will caused salting out process and formed protein precipitate. This
precipitation do not change the structure of protein. (Sinaga, Masdalena,)
 Reaction 4.9 Precipitation of Ammonium Sulphate

b. Precipitation by Concentrated Mineral Acid

Mineral acid conditioned on protein can caused the form of salt compound
from acid reaction of amino protein group. Another factor can caused irreversible
denaturation and protein precipitation. But commonly formed reversible precipitate
when adding concentrated mineral acid (except HNO3). (Hidajati, Nurul, 2017)
c. Precipitation by heavy metal
The principal of this precipitation is netralize load. The presence of positive
load from heavy metal will netralize ion of proteine and formed neutral proteinate
salt precipitated. The properties of this reaction is reversible. It will be solute again
when added alkali such as NH3 and NaOH (Hidajati, Nurul, 2017)

Reaction 4.10 Precipitation by heavy metal

Protein Hydrolisys
 Protein hydrolysis is a method used to obtain protein hydrolysate. Hydrolysis of
proteins is influenced by the concentration of hydrolysis materials, substrate
concentration, temperature, pH and time (Hidayat, 2005). Proteins undergoing hydrolytic
degradation with acids, bases, or proteolytic enzymes that produce products of amino
acids and peptides are called protein hydrolyzates (Kurniawan et al., 2012).The use of
enzymes in hydrolyzing proteins is considered to be the safest and most beneficial. This
is due to the enzyme's ability to hydrolyze proteins to produce hydrolyzate products that
avoid changes and damage to products (Johnson and Peterson, 1974 in Kurniawan et al.,
2012).

Acid Hydrolisys

Hydrolysis by using an inorganic strong acid, such as concentrated HCl or H2SO4

(4-8 normal), is then heated at a boiling temperature or can be carried out with pressure
over one atmosphere, for several hours. Side effects that occur with acid hydrolysis is the
destruction of some amino acids (tryptophan, some serine and threonin).

Reaction 4.11 Hdrolysis of protein (Fessenden, 1982)

Base Hydrolisys

Protein hydrolysis using a base is a polypeptide-breaking process by using a

strong alkali, such as NaOH and KOH at high temperatures, for several hours, with
pressure above one atmosphere. However serin and threonin are damaged by a base.

Sulphur identification

Sulphur identification showing positive result on amino acid which contain amino
acid that have sulphur group, such as sistein, sistin and mentionin. The procedure are,
protein solution and concentrated NaOH heated, then added Pb-acetic. If the protein
contain sulphur, it will formed black precipitate, Sulphur sulphida (PbS). (Syabana, 2011)
 Pb(CH3COO)2 (aq) + 4 NaOH(aq) → Na2PbO2 (aq)

Na2PbO2(aq) + 2S2- (aq) + 2H2O(l) → PbS(s) +2 NaOH(aq) +2OH-(aq)

Reaction 4. 12 Sulphur Identification

Protein Denaturation

Denaturation is a protein that loose its biological properties. (Fessenden, 1987)

Protein denaturation is the changing structure of secondary, tertiary, and kuarterner chain
without changing the primary structure (without cut the peptide bond). (Himelia
Labiratories, no year) Anything that breaks the bonds maintaning the three-dimensional
shape of the protein will cause the protein to denature (unfold). Because these bonds are
weak, proteins are easily denaturated. The totally random conformation of a denaturated
protein is called random coil. (Bruice, Paula Yurkani, 2011)

Picture 4.5 Denaturation (Himelia Labiratories, no year)

Some ways that caused denaturation of protein :

 Changing the pH. Because it changes the charge on many of the side chains. This
disrupt electrostatic attractions and hydrogen bonds.
 Adding reagent such as urea or guanidine hydrochloride caused the formation of
hydrogen bonds to protein groups that are stronger than teh hydrogen formed
between the groups.
  Detergent such as sodium dodecyl sulfate denature proteins by associating with the
nonpolar groups of the protein, similar as normal hydrophobic interractions.
 Oragnic solvent can caused denaturation with disrupting hydrophobic interactions.
 Protein also denaturated by heating or agitation. It disrupt the attractive force by
increasing the molecular motion. The common example is the change that occurs to
the white of an egg when it is heated or whipped.

V. Tools and Materials

Tools :

- Test tube 50 pieces

- Beaker glass(50 mL,100 mL, 250 mL) 3 pieces
- Bunsen bunner 1 pieces
- Spatula 1 piece
- Meassurement glass 2 pieces
Materials :
- Protein solution (egg and milk) sufficient
- Acetate acid sufficient
- (NH4)2SO4 solution sufficient
- HNO3 sufficient
- CuSO4 sufficient
- PbSO4 sufficient
- ZnSO4 sufficient
- HgSO4 sufficient
- NaNO2 sufficient
- Congo indicator sufficient
- PP indicator sufficient
- Ninhydrin solution sufficient
- Ammoniu sulphate sufficient
-
 VI. Lanes Work
Denaturation of Protein
a. Denaturation because of acetate addition

5 mL Protein Solution

- Entered into test

tube
- Added 2 drops
acetate acid 1 N
- Shaken it, flake
will be formed
- Heated in steam
bath for 5 minutes
- Observed the
precipitate changes

Result

b. Denaturation because of heating

2-3 mL Protein Solution

- Entered into test tube

- Heated for 1 minute
(precipitate will be
formed)
- Cooled
- Divided into two tubes

Test tube 1 Test tube 2

- Added 1-2 - Heated it

drops of
(NH4)2SO4
- Heated it

Result Result
 c. Denaturation because of Formaldehyde

1-15 mL formaldehyde 2 mL Aquades

- Entered into
test tube
- Added
protein
solution
drop by
drop
- Observed
Result

2. Amfoter characteristic of Protein

- test in acid condition

3 mL of Aquadest

- Entered into test

tube
- Added 1 drop of
HCl
- Added several
drops of Congo
indicator
- Added 2-3 mL of
protein solution
- Noted the color
changing

Result
- Test in base condition

3 mL of NaOH 0,1 M

- Entered into test

tube
- Added several
drops of PP
indicator

Result

2-3 mL protein solution

- Entered into test

tube
- Added drop by
drop NaOH 0,1
M
- observed

Result

Precipitation of Protein

a. Precipitation of protein with Ammonium Sulphate

3-4 mL of protein

- Poured into test tube

- Added 3-4 mL saturated
Ammonium Sulphate
- Shaken it slowly (the solution
become turbid)
- Added 2-3 mL aquadest
- Shaken it again

Result
b. Protein precipitation with mineral acid

1 mL of HNO3

- Poured into test tube

- Tilt the test tube
- Added 1-1,5 mL protein
solution drop by drop the
wall
- Let it stand until the white
ring formed as protein
precipitate
- Shaken it
- Added HNO3, until formed
white precipitate

Result

1-2 mL of concentrated HCl

- Poured into test tube

- Tilted the tube
- Added 1-1,5 mL protein solution
- Taken up the tube again
- Let it stand until white ring
formed as protein precipitate
- Shaken it
- Added HCl again, formed clear
solution

Result
c. Protein precipitation with heavy metal

1-1,5 mL Protein Solution 1-1,5 mL Protein

Solution
- Poured into test tube - Poured into test tube
- Added drop by drop - Added drop by drop
CuSO4 ZnSO4
- Shaken it - Shaken it
Blue precipitate
Blue precipitate
- Added CuSO4 drop
- Added ZnSO4 drop
by drop
by drop
- Shaken until
- Shaken until
precipitate dilute
precipitate dilute
- Observed the
- Observed the
changing process
changing process
Result
Result

1-1,5 mL Protein 1-1,5 mL Protein Solution

Solution
- Poured into test - Poured into test
tube tube
- Added drop by drop - Added drop by drop
FeSO4 HgSO4
- Shaken it - Shaken it
Blue precipitate
Blue precipitate
- Added FeSO4 drop
by drop
- Added HgSO4 drop
- Shaken until
by drop
precipitate dilute
- Shaken until
- Observed the
precipitate dilute
changing process
- Observed the
Result changing process

Result
4. Reaction the color Protein

1. Biuret Reaction

3 mL of Protein solution

- Poured into test

tube
- Added 1 mL of
concentrated
HNO3
- Heated until
formed yellow
solution
- Cooled it
- Added ammonia
until formed
orange solution

Result

2. Xanthoprotein

3 mL of Protein solution

- Poured into test

tube
- Added 1 mL of
concentrated
HNO3
- Heated until
formed yellow
solution
- Cooled it
- Added ammonia
until formed
orange solution

Result
3. Ninhydrin reaction

Protein solution 5%

- Set pH=7
- Taken from this solution
- Added 10 drops of
ninhydrin 0,2% solution
- Heated in 1000C for 10
minutes
- Observed the changes

Result

4. Millon reaction

2 mL of Protein solution

- Poured into test

tube
- Added 1 mL
Mercurysulphate
reagent (Millon
reagent)
- Heated it, formed
yellow solution
- Cooled using cold
water
- Added 1 drop of 1%
NaNO2 solution
- Heated it again.
Formed red solution

Result
5. Hopkin-cole Reaction

1 mL of Protein solution

- Poured into test

tube
- Added 1 drop of
formaldehyde
- Added 1 drop of
mercurysulphate
reagent
- Added 1 mL H2SO4
(concentrated)
through the wall
tube until formed
two layers. There is
purple ring. If it is
shaken the solution
become purple

Result

5.Protein hydrolisis and sulphur identification

1 mL of protein solution

- Added 1 mL of
NaOH 40%
- Heated for 1
minute
- Added 1 drop of
Pb-acetic formed
black solution
(indicate PbS)

Result
 VII. Obseravtion Result

rocedure Observation Result Reaction/assumption Conclusion

 Denaturation of Protein Before: After: The form of
c. Denaturation because of acetate precipitate on the
addition White egg: Egg + acetate tube, indicate that
White solution acid: form acetate acid can
white cause the
Aquadest: precipitate on denaturation of
5 mL Protein Solution colorless the bottom of protein
the tube
- Entered into test Acetate acid:
tube colorless Milk +
- Added 2 drops acetate acid:
acetate acid 1 N Formaldehyde the milk
- Shaken it, flake : colorless solution
become solid
1. will be formed
Milk: white on the bottom
- Heated in steam
solution of the tube
bath for 5 minutes
- Observed the
precipitate changes

Result
 Egg tube I: The heating
d. Denaturation because of heating the solution process can cause
become white the denaturation
2-3 mL Protein of protein.
Egg tube II: Indicated by the
Solution
the color form of precipitate
- Entered into
become white on the bottom of
test tube
and form the tube.
- Heated for 1 solid
minute precipitate
(precipitate
will be formed) Milk tube I:
- Cooled the color
- Divided into become white
two tubes
Milk tube II:
the color
become white
Test Test
tube 1 tube 2
- Added 1-2 drops- Heate
of (NH4)2SO4 d it
- Heate d it

Result Result
c. Denaturation because of Egg: there is The adding of
Formaldehyde turbid formaldehyde in
precipitate on the tube can cause
the upper the denaturation
1-15 mL formaldehyde 2 mL Aquades layer of the of protein,
tube indicated by the
form of precipitate
Milk: there is on each tube.
white
precipitate on
- Entered into the upper
test tube layer of the
- Added tube
protein
solution
drop by
drop
- Observed
the
precipitate

Result
 Amfoter characteristic of Protein Aquadest: Aquadest + From this
- test in acid condition colorless HCl + Congo experiment we
indicator: can conclude that
3 mL of Aquadest HCl 1 N: light blue protein can react
colorless with acid and base
- Entered into test Aquadest + indicated by the
tube Congo HCl + Congo color change.
Indicator: indicator + - In acid
- Added 1 drop of
orange solution milk: white condition:
HCl pink Egg change
- Added several from colorless
drops of Congo to light pink.
indicator Milk change
- Added 2-3 mL of from white to
protein solution white pink.
2. - Noted the color
changing

Result
- Test in base condition NaOH 0,1 M: Aquadest + - In base
colorless NaOH + PP condition: egg
3 mL of NaOH 0,1 M solution: pink change from
PP indicator: solution colorless into
pink solution pink solution.
- Entered into test Aquadest + Milk change
tube NaOH + from white to
- Added several milk: pink white pink
drops of PP white solution solution.
indicator
Aquadest +
Result NaOH + PP +
Egg: pink
solution

2-3 mL protein solution

- Entered into test

tube
- Added drop by
drop NaOH 0,1
M
- observed

Result
 Precipitation of Protein Arab egg: Milk + Precipitate of
d. Precipitation of protein with colorless (NH4)2SO4: protein dissolved
Ammonium Sulphate white solution when added with
Milk: white Shaken: white aquadest. Indicate
solution solution that reaction
3-4 mL of protein +aquadest: between protein
Saturated white solution and (NH4)2SO4 is
- Poured into test (NH4)2SO4: + white reversible reaction
tube colorless precipitate
- Added 3-4 mL solution egg +
saturated (NH4)2SO4:
Ammonium Aquadest: white solution
Sulphate colorless Shaken:
- Shaken it slowly solution yellow
(the solution solution +=
3. become turbid) white
precipitate +
- Added 2-3 mL
aquadest:
aquadest
colorless
- Shaken it again solution
Result
e. Protein precipitation with HNO3: HNO3 + Reaction of
mineral acid colorless Milk: white protein with
solution ring HNO3 is
Upper: white reversible reaction
1 mL of HNO3 solution indicated by the
Lower: formation of
- Poured into test colorless precipitate when
tube solution added saturated
- Tilt the test tube Shaken: HNO3. Meanwhile
- Added 1-1,5 mL yellow reaction of HCl
protein solution solution and with protein is
drop by drop the white reversible reaction
wall precipitate indicated by the
- Let it stand until + HNO3: precipitation
the white ring yellow dissolved when
solution added saturated
formed as
HNO3 + egg: HCl.
protein whitening
precipitate Upper: white
- Shaken it solution
- Added HNO3, Lower:
until formed colorless
white precipitate Shaken:
yellow
Result solution+
white
precipitate
+HNO3:
yellow
solution +
yellow
precipitate
 HCl: colorless HCl+milk=
solution white solution
Shaken:
1-2 mL of concentrated HCl
turbid
+HCl:
- Poured into test colorless
tube solution
- Tilted the tube HCl+ egg:
- Added 1-1,5 mL white ring
protein solution Shaken:
- Taken up the turbid
tube again +HCl: turbid
- Let it stand until
white ring
formed as
protein
precipitate
- Shaken it
- Added HCl
again, formed
clear solution

Result
f. Protein precipitation with heavy ZnSO4: Milk Reaction of
metal colorless +CuSO4: blue protein with heavy
solution solution metals (ZnSO4,
1-1,5 mL Protein Solution CuSO4: blue Shaken:blue CuSO4, FeSO4,
solution precipitate PbSO4, HgSO4)
- Poured into test tube FeSO4: orange +CuSO4: blue are reversible
- Added drop by drop solution solution + reaction, indicated
CuSO4 HgSO4: millon blue by the
- Shaken it reagent precipitate precipitation
Colorless dissolved when
Blue precipitate solution Egg+CuSO4: added saturated
PbSO4: blue heavy metals
- Added CuSO4 drop colorless precipitate (ZnSO4, CuSO4,
by drop solution Shaken: blue FeSO4, PbSO4,
precipitate HgSO4)
- Shaken until
+CuSO4: blue
precipitate dilute
solution
- Observed the
changing process
Result

1-1,5 mL Protein
Solution
- Poured into test tube
- Added drop by drop
ZnSO4
- Shaken it
 Milk:
+ZnSO4:
white solution
Shaken: white
precipitate
+ZnSO4:
white
precipitate

1-1,5 mL Protein
Solution
- Poured into test
tube
- Added drop by drop
FeSO4
Egg
+FeSO4:
yellow
solution
+FeSO4: blue
precipitate
Shaken:
yellow
precipitate
+FeSO4:
yellow
solution

Milk
+FeSO4: grey
precipitate
Shaken: grey
precipitate
 Egg:
1-1,5 mL Protein Solution +HgSO4:
white
precipitate
- Poured into test
Shaken: white
tube
precipitate
- Added drop by drop +HgSO4:
HgSO4 colorless
- Shaken it solution+
white globe
Blue precipitate

- Added HgSO4 drop

by drop
- Shaken until
precipitate dilute
- Observed the
changing process

Result
 PbSO4+egg:
1-1,5 mL Protein colorless
Solution solution
- Poured into test tube Shaken:
- Added drop by drop colorless
PbSO4 solution
- Shaken it +PbSO4:
colorless
Blue precipitate solution

PbSO4+milk:
- Added PbSO4 drop white
by drop precipitate
- Shaken until Shaken: white
precipitate dilute precipitate
- Observed the +PbSO4:
changing process white solution
+ white
Result precipitate
 Reaction the color Protein Milk: white Milk + The conclusion is
solution NaOH: white there are peptides
6. Biuret Reaction solution bond in egg and
Egg: colorless milk indicated by
solution Milk+NaOH+ form of purple
3 mL of Protein solution CuSO4 0,5%: complex.
NaOH: purple
- Poured into test colorless solution
tube solution
- Added 1 mL of Egg+NaOH:
40% NaOH CuSO4 0,5%: colorless
- Added drop by light blue solution
drop 0,5% CuSO4, solution
the color become Egg+NaOH+
red or purple CuSO4 0,5%:
purple
Blue precipitate solution
4.

7. Xanthoprotein HNO3 Milk+HNO3:

 concentrated: white
colorless yellowish From this
solution solution experiment, we
3 mL of Protein solution can conclude that
Ammonia: Egg+HNO3: in egg and milk
- Poured into test colorless light there are benzene
solution yellowish group (phenyl
tube
solution ring) indicated by
- Added 1 mL of
form of orange
concentrated Milk+HNO3+ solution.
HNO3 heated: white
- Heated until yellowish
formed yellow solution
solution
- Cooled it Egg+HNO3 +
- Added ammonia heated: white
until formed yellowish
orange solution solution

Result Milk+ HNO3

+ heated +
ammonia:
orange
solution

Egg+HNO3 +
heated +
ammonia:
orange
solution
8. Ninhydrin reaction Ninhydrin: Milk+ From this
colorless ninhydrin: experiment we
Protein solution 5% white solution can conclude that
in egg and milk
- Set pH=7 Egg+ contain α-amino
- Taken from this ninhydrin: acid indicated by
solution colorless purple solution
- Added 10 drops of solution formed
ninhydrin 0,2%
solution Milk+
ninhydrin +
- Heated in 1000C
heated: purple
for 10 minutes solution
- Observed the
changes Egg+
ninhydrin +
Result heated: purple
solution
 9. Millon reaction Egg: colorless Egg+ millon From this
solution + NaNO2 + experiment we
heated: dark can conclude that
2 mL of Protein solution
Milk: white purple in egg and milk
solution precipitate, on not contain tirosin
- Poured into test top tube + indicated by not
tube yellowish formed red
- Added 1 mL solution complex
Mercurysulphate precipitate.
reagent (Millon Milk + millon
reagent) + NaNO2 +
- Heated it, formed heated: milk
yellow solution precipitate’s
- Cooled using cold color is
water brown and
orange
- Added 1 drop of 1%
solution
NaNO2 solution
- Heated it again.
Formed red solution

Result
10. Hopkin-cole Reaction Milk: white Milk + The form of
solution formaldehyde purple ring
+ millon precipitate
Egg: colorless reagent + HCl indicated that
1 mL of Protein solution solution (concentrated) there is tryptophan
: form 2 compound in the
- Poured into test layers there milk and egg
tube are brown and
- Added 1 drop of dark purple
formaldehyde ring on the
- Added 1 drop of bottom tube
mercurysulphate
reagent Egg+
- Added 1 mL H2SO4 formaldehyde
+ millon
(concentrated)
reagent + HCl
through the wall
(concentrated)
tube until formed : purple ring
two layers. There is on bottom
purple ring. If it is tube
shaken the solution
become purple

Result
 Protein hydrolisis and sulphur Milk: white Milk + NaOH The use of NaOH
identification solution 40% + Pb- is for cutting the
Acetate: dark protein bond, and
1 mL of protein solution Egg: colorless brown thecolor change
solution solution become brown,
indicated that
- Added 1 mL of
NaOH: Egg+ NaOH there Sulphur
NaOH 40% colorless 40% + Pb- compound in the
- Heated for 1 solution Acetate: dark protein (milk).
minute red solution
- Added 1 drop of
Pb-acetic formed
black solution
(indicate PbS) Pb(CH3COO)2 (aq) + 4
Result NaOH(aq) → Na2PbO2 (aq)
5.
Na2PbO2(aq) + 2S2- (aq) +
2H2O(l) → PbS(s) +2 NaOH(aq)
+2OH-(aq)
VIII. Anylisis and Explanation

1. Denaturation of Protein
a. Denaturation because of acetic acid, heat and formaldehyde

5 mL protein solution, poured into test tube and added with 2 drops of
acetate acid. Then the tube is shaken and observed the changing. It will form white
precipitate, indicate that there is reaction between protein from egg or from milk
with acetic acid. Both of egg and milk, when reacted with acetic acid will form white
precipitate. It is sign of protein denaturation. After form white precipitate from egg
and milk, then both tube is heated, observe the change. The precipitation of
protein will be more concentrated. Because the heating process of reaction will
increase the reaction rate of reaction.

Before conducting the experiment, firstly the egg is taken the white phase.
The yellow phase is saved. The reason of choosing the white of egg, is because the
with egg is contain more protein than the yellow egg. It means the reaction of
protein will conduct correctly.

The reason why choosing the milk, is because milk contain of protein from
animals. At least 3,4 grams of protein contain from 100 grams of milk. Protein (from
the Greek protas meaning "of primary importance") is a complex, high-molecular-
weight organic compound that consists of amino acids joined by peptide
bonds. Proteins are natural polymer molecules consisting of amino acid units. The
number of amino acids in proteins may range from two to several thousand.

Proteins are probably the most important class of biochemical molecules,

although of course lipids and carbohydrates are also essential for life. Proteins are
the basis for the major structural components of animal and human tissue.

Proteins are essential to the structure and function of all living cells and
viruses. Many proteins are enzymes or subunits of enzymes, catalyzing
chemical reactions. Other proteins play structural or mechanical roles, such as those
 that form the struts and joints of the cytoskeleton, serving as biological scaffolds
for the mechanical integrity and tissue signaling functions. Proteins can be
hydrolyzed by acids, bases or specific enzymes.

Denaturated proteins are characterized by:

1-Loss of function: Most biological proteins lose their biological function when
denatured, for example, enzymes lose their catalytic activity.

2- They become less soluble. As a result, they are easily precipitated.

3- Reversibility and irreversibility: In many proteins (unlike egg whites),

denaturation is reversible (the proteins can regain their native state when the
denaturing influence is removed).

Picture a.1 denaturation of protein mechanism

2. Amfoter Charateristic of Protein

The purpose of this experiments is to identify the amfoter charateristic of protein, react in
acid condition and base condition. Protein can react with acid and base beacuse the structure
of protein are two groups, NH3+ react with base and COO- react with acid. (Fessenden,1986)
√ Egg sample

First, in acid condition added 3 mL of aquadest into test tube, then added 1 mL of HCl 1 N.
after that added several drops of congo indicator. Aquadest and HCl are colourless solution and
congo indicator is orange solution. When aquadest, HCl and congo indicator are mixed, form
light purple solution. The function of adding congo indicator to identify the solution that in acid
condition. Possitive acid when the colour of solution is blue/purple. Used congo indicator
beacuse congo is indicator of acid with range of pH is 3,8-5,4. After that added protein solution
is that egg solution into test tube. Egg solution is colourless and the result after adding egg
solution is light pink solution. The changed of colour indicate that the sample is react.

The structure of protein contain combination of amino acid arrangement in carbonyl group
and amino acid with peptide bond. The colour changed is caused by concentration of H + is high
can bonding with –COO- formed –COOH. In acid condition protein molecule formed positive ion.
(Anonym,2015)

The reaction protein in acid condition as below :

In base condition, added 3 mL of aquadest into test tube, then added NaOH 0,1 M into test
tube. After that added several drops of PP indicator. Aquadest, NaOH and PP indicator is
colourless solution. After adding PP indicator the solution chenged into pink solution. The
function of adding PP indicator to test that in solution is base condition and to test the protein
solution can react with base or not. If the solution is base, the pink solution is not change..
Then, added 2 mL egg solution into test tube. The result is pink solution, it means in protein can
react with base.
 In base condition, the colour changed to pink is caused by concentration of OH - is high can
bonding with ions H+ with in NH3+ group. In base condition protein molecule formed negative
ion. (Anonym,2015)

The reaction protein in base condition as below :

√Milk sample

First, in acid condition added 3 mL of aquadest into test tube, then added 1 mL of HCl 1 N.
after that added several drops of congo indicator. Aquadest and HCl are colourless solution and
congo indicator is orange solution. When aquadest, HCl and congo indicator are mixed, form
light purple solution. After that added protein solution is that milk solution into test tube. Milk
solution is white solution and the result after addingmilk solution is white pink solution. The
changed of colour indicate that the sample is react.

The structure of protein contain combination of amino acid arrangement in carbonyl group
and amino acid with peptide bond. The colour changed is caused by concentration of H + is high
can bonding with –COO- formed –COOH. In acid condition protein molecule formed positive ion.
(Anonym,2015)

The reaction protein in acid condition as below :

 In base condition, added 3 mL of aquadest into test tube, then added NaOH 0,1 M into test
tube. After that added several drops of PP indicator. Aquadest, NaOH and PP indicator is
colourless solution. After adding PP indicator the solution chenged into pink solution. The
function of adding PP indicator to test that in solution is base condition and to test the protein
solution can react with base or not. If react the pink solution is not change. Then, added 2 mL
milk solution into test tube. The result is pink solution, it means in protein can react with base.

In base condition, the colour changed to pink is caused by concentration of OH- is high can
bonding with ions H+ with in NH3+ group. In base condition protein molecule formed negative
ion. (Anonym,2015)

The reaction protein in base condition as below :

3. Protein precipitation

The purpose of this third experiment is to understand the cause of precipitation process
in protein. The precipitation divided into three sub group there are, precipitation of protein
with ammonium sulphate, protein precipitation with mineral acid and the last is protein
precipitation with heavy metal. There are two kind of protein solution that we used on this
experiment. The two protein solution are yolk of ‘arab chicken and fresh milk.

1) Precipitation of protein with ammonium sulphate

The purpose of this experiment is to observed the precipitation of protein when

added ammoium sulphate. The first step is 3 ml of fresh milk entered into test tube,
then added 3 ml of saturated ammonium sulphate. It will formed white solution, then
the solution shaken until formed white precipitate. Then added 2 – 3m of aquadest.
After added aquadest, the solution formed larger number of white precipitate. The raw
colour of milk is white, and the colour of ammonium sulphate is colourless. The
precipitation of protein happened because ammonium sulphate is decrease the
solubility of proteins in to water because of water adsorbed by salt. Ammonium
sulphate that soluted into water will caused salting out process and formed protein
precipitate. When added aquadest the number of solution is bigger because there are a
lot number of water ion that adsorbed by salt and make protein less solute in water
caused the number of white precipitation larger.

And then for the experiment using yolk of ‘arab’ chicken as the protein solution.
Done with the same steps. The first step is 3 ml of yolk entered into test tube, then
added 3 ml of saturated ammonium sulphate. It will formed turbid solution, then the
solution shaken until formed white precipitate in turbid solution. Then added 2 – 3 mL
of aquadest. After added aquadest, the solution formed larger number of white
precipitate. The raw colour of yolk is colourless, and the colour of ammonium sulphate
is colourless. The precipitation of protein happened because ammonium sulphate is
decrease the solubility of proteins in to water because of water adsorbed by salt.
Ammonium sulphate that soluted into water will caused salting out process and formed
protein precipitate. When added aquadest the number of solution is bigger because
there are a lot number of water ion that adsorbed by salt and make protein less solute
in water caused the number of white precipitation larger. The reaction is,
2) Precipitation of protein with mineral acid
The purpose of this experiment is to differentiate the characteristic solubillity of
protein’s reversibly and irreversibly. Two kind of concentrated acid that used on this
experiment are chloride acid and nitric acid. The principal of this experiment is protein
solution will formed reversible precipitation when added mineral acid, except HNO3 as a
mineral acid. First is using chloride acid as the concentrated mineral acid. Concentrated
Cloride acid is colourless solution with purgent odor. to reacted this concentrated acid
have to doing in a fume hood. The steps of experiment are 3 ml of HCl entered into test
tube, then tilted the tube, after that added 1 ml of fresh milk drop by drop of through the
tube wall. The purpose of this manner is the white ring formed when the tube let on
stand. This white ring is the precipitate of protein solution. Then shaken it and the
solution formed white precipitate, on white solution. Then added HCl drop by drop. Until
the solution become colourless. When HCl added by fresh milk protein denaturate
without broken the formation of its protein structure, then when HCl added saturatedly.
The protein solute because the precipitate formed the HCl and protein again or it called
reversible denaturation.
Done the same steps on the experiment with yolk of ‘arab’ chicken as the protein
solution. 3 ml of HCl entered into test tube, then tilted the tube, after that added 1 ml of
yolk drop by drop of through the tube wall. The purpose of this manner is the white ring
formed when the tube let on stand. This white ring is the precipitate of protein solution.
Then shaken it and the solution become turbid. Then added HCl drop by drop. Until the
solution become colourless. When HCl added by yolkm the protein denaturated without
broken the formation of its structure, then when HCl added saturatedly. The protein
solute because the precipitate formed the HCl and protein again or it called reversible
denaturation. But from our experiment, the yolk solution is not colourless but turbid
solution without a precipitation. It indicated the reaction is reversible but not the perfect
one, because the volume of yolk is 1 ml so, needed a lot number of HCl so the solution
can become colourless. The reaction is,

After that using concentrated HNO3 as the mineral acid. First, using fresh milk as
the protein solution. HNO3 is colourless solution with purgent odor. The steps of
experiment are 3 ml of HNO3 entered into test tube, then tilted the tube, after that added 1
ml of fresh milk drop by drop through the tube wall. The purpose of this manner is the
white ring formed when the tube let on stand. This white ring is the precipitate of protein
solution. Then shaken it and the solution formed white precipitate, on white solution.
Then added HNO3 drop by drop. The colur of solution and precipitate changing from
white into yellow. When HNO3 added by fresh milk protein precipitated without broken
the formation of its protein structure, then when HNO3 added saturatedly. The protein
denaturated irreversibly this same with the theory, HNO3 is mineral acid that can not
formed reversibly precipitation when added saturatedly.
The second is using yolk of ‘arab’ chicken as the protein solution. HNO3 is
colourless solution with purgent odor. The steps of experiment are 3 ml of HNO3 entered
into test tube, then tilted the tube, after that added 1 ml of yolk drop by drop through the
tube wall. The purpose of this manner is the white ring formed when the tube let on
stand. This white ring is the precipitate of protein solution. Then shaken it and the
solution become turbid with white precipitate. Then added HNO3 drop by drop. The
colour of solution and precipitate changing from white into yellow. When HNO3 added
by yolk, protein precipitated without broken the formation of its protein structure, then
when HNO3 added saturatedly. The protein denaturated irreversibly this same with the
 theory, HNO3 is mineral acid that can not formed reversibly precipitation when added
saturatedly. The reaction is,

3) Precipitation of heavy metals

The purpose of this experiment is to identified the precipitation of protein because
of heavy metals. There are five kind of heavy metals that used on this experiment,
CuSO4, ZnSO4, PbSO4, FeSO4 and HgSO4. The principal of this precipitation is netralize
load. The presence of positive load from heavy metal will netralize ion of proteine and
formed neutral proteinate salt precipitated. The properties of this reaction is reversible. It
will be solute again when added alkali such as NH3 and NaOH
The first is precipitation of fresh milk with blue solution of CuSO4. 1 ml of fresh
milk entered into test tube. Then added CuSO4 drop by drop unti formed blue precipitate
after that shaken it. The solution and precipitate become blue. Then added drop by drop
of CuSO4, shaken it until the precipitate diluted. So the solution become blue without a
precipitate. CuSO4 with 2- as its anion netralize the ion of protein and fomr Cu-complex,
indicated by the blue precipitate. But when added saturated CuSO4 the protein precipitate
diluted and Cu-complex apart to be anion Cu and kation of protein.
Then experiment of yolk with blue solution of CuSO4. 1 ml of yolk entered into
test tube. Then added CuSO4 drop by drop unti formed blue precipitate after that shaken
it. The solution and precipitate become blue. The number of precipitate on this protein
solution are not bigger than on fresh milk. Then added drop by drop of CuSO4, shaken it
until the precipitate diluted. So the solution become blue without a precipitate. CuSO4
with -2 as its anion netralize the ion of protein and form Cu-complex, indicated by the
blue precipitate. But when added saturated CuSO4 the protein precipitate diluted and Cu-
complex apart to be anion Cu and kation of protein. The reaction is,
 The second is precipitation of fresh milk with colourless solution of ZnSO4. 1 ml
of fresh milk entered into test tube. Then added ZnSO4 drop by drop until formed white
precipitate, after that shaken it. The solution and precipitate become white. Then added
drop by drop of ZnSO4, shaken it until the precipitate diluted. So the solution become
turbid without a precipitate. ZnSO4 with 2- as its anion netralize the ion of protein and
fomr Zn-complex, indicated by the white precipitate. But when added saturated ZnSO4
the protein precipitate diluted and Zn-complex apart to be anion Zn and kation of protein.
But from our experiment the final result is white solution and white precipitate. It is
because when added ZnSO4 the protein solution not diluted.
Then experiment of yolk with colourless solution of ZnSO4. 1 ml of yolk entered
into test tube. Then added ZnSO4 drop by drop until formed white precipitate after that
shaken it. The solution turbid with white precipitate. The number of precipitate on this
protein solution are not bigger than on fresh milk. Then added drop by drop of ZnSO 4,
shaken it until the precipitate diluted. So the solution become turbid without precipitate.
ZnSO4 with -2 as its anion netralize the ion of protein and form Zn-complex, indicated by
the white precipitate. But when added saturated ZnSO4 the protein precipitate diluted and
Zn-complex apart to be anion Zn and kation of protein. The reaction is,

The third is precipitation of fresh milk with orange solution of FeSO4. 1 ml of

fresh milk entered into test tube. Then added FeSO4 drop by drop until formed white
precipitate, after that shaken it. The solution is grey and the precipitate is white. Then
added drop by drop of FeSO4, shaken it until the precipitate diluted. So the color of
solution is grey brownish without a precipitate. FeSO4 with 2- as its anion netralize the
ion of protein and fomr Fe-complex, indicated by the white precipitate. But when added
saturated FeSO4 the protein precipitate diluted and Fe-complex apart to be anion Fe and
kation of protein.
Then experiment of yolk with orange solution of FeSO4. 1 ml of yolk entered into
test tube. Then added FeSO4 drop by drop until formed yellow precipitate after that
shaken it. The solution becomes yellow and the precipitate is orange. Then added drop by
drop of FeSO4, shaken it until the precipitate diluted. So the solution become yellow
without precipitate. FeSO4 with -2 as its anion netralize the ion of protein and form Fe-
complex, indicated by the orange precipitate. But when added saturated FeSO4 the
protein precipitate diluted and Fe-complex apart to be anion Fe and kation of protein. The
reaction is,

The fourth is precipitation of fresh milk with colourless solution of HgSO4. 1 ml

of fresh milk entered into test tube. Then added HgSO4 drop by drop until formed white
precipitate, after that shaken it. The solution and precipitate are white. Then added drop
by drop of HgSO4, shaken it until the precipitate diluted. So the color of solution is
colourless with white glob. HgSO4 with 2- as its anion netralize the ion of protein and
fomr Hg-complex, indicated by the white precipitate. But when added saturated HgSO4
the protein precipitate diluted and Hg-complex apart to be anion Hg and kation of
protein.
Then experiment of yolk with colourless solution of HgSO4. 1 ml of yolk entered
into test tube. Then added HgSO4 drop by drop until formed white precipitate after that
shaken it. The solution and precipitate are white. Then added drop by drop of HgSO4,
shaken it until the precipitate diluted. So the solution become colourless with white golb.
HgSO4 with -2 as its anion netralize the ion of protein and form Hg-complex, indicated
by the white precipitate. But when added saturated HgSO4 the protein precipitate diluted
and Hg-complex apart to be anion Hg and kation of protein. The reaction is,

The fifth is precipitation of fresh milk with colourless solution of PbSO4. 1 ml of

fresh milk entered into test tube. Then added PbSO4 drop by drop until formed white
precipitate, after that shaken it. The solution and precipitate are white. Then added drop
by drop of PbSO4, shaken it until the precipitate diluted. So the color of solution is white
without precipitate. PbSO4 with 2- as its anion netralize the ion of protein and form Pb-
complex, indicated by the white precipitate. But when added saturated PbSO4 the protein
precipitate diluted and Pb-complex apart to be anion Pb and kation of protein.
Then experiment of yolk with colourless solution of PbSO4. 1 ml of yolk entered
into test tube. Then added PbSO4 drop by drop until formed white precipitate after that
shaken it. The solution is turbid. Then added drop by drop of PbSO4, shaken it until the
precipitate diluted. So the solution become colourles. PbSO4 with -2 as its anion netralize
the ion of protein and form Pb-complex, indicated by the white precipitate. But when
added saturated PbSO4 the protein precipitate diluted and Pb-complex apart to be anion
Pb and kation of protein. The reaction is,
4. Colour Reaction of Protein

a. Biuret test

The purpose of this experiment is to identify the presence of protein that is peptide bond in
sample(egg and milk solution). The biuret reagent can made by reacting NaOH with CuSO4
(Ernalia,2015)

 Egg
First, added 3 mL of egg solution into test tube, then added 1 mL NaOH 40%. The
function of adding NaOH to alkali condition. After added drop by drop CuSO4 0,5% until the
solution purple/red. NaOH is colourless solution and CuSO4 is light blue solution. The result of
this experiment is purple solution, indicate that there are two more peptide bond in egg
sample. If there are dipeptide the result is blue solution and if there are of complex peptide
bond the colour is red. (Vina, 2015)

The purple solution formed because the complex compound are formed between Cu 2+
with –CO group and NH from peptide molecule bond in base condition. The alkaline CuO 4
solution reacts with the polypeptide, while the polypeptide is the protein constituent.
Indicating the presence of proteins is more peptide bonding, it is evident when the addition of
CuSO4 solution and shaken the solution remain purple indicating that the peptide bond is
strong, because if the peptide bond is weak when the protein solution is added CuSO4 solution,
the purple color fades when shaken. (Vina,2015)

 Milk

First, added 3 mL of milk solution into test tube, then added 1 mL NaOH 40%. The function
of adding NaOH to alkali condition. After added drop by drop CuSO4 0,5% until the solution
purple/red. NaOH is colourless solution and CuSO4 is light blue solution. The result of this
experiment is purple solution, indicate that there are two more peptide bond in egg sample. If
there are dipeptide the result is blue solution and if there are of complex peptide bond the
colour is red. (Vina, 2015)

The purple solution formed because the complex compound are formed between Cu 2+
with –CO group and NH from peptide molecule bond in base condition. The alkaline CuSO4
solution reacts with the polypeptide, while the polypeptide is the protein constituent.
Indicating the presence of proteins is more peptide bonding, it is evident when the addition of
CuSO4 solution and shaken the solution remain purple indicating that the peptide bond is
strong, because if the peptide bond is weak when the protein solution is added CuSO 4 solution,
the purple color fades when shaken. (Vina,2015)

This reaction as bellow:

OH OH

+ NaOH + CuSO4 Na2SO4 + H2O +

H 2C H 2C H 2C

HC NH 2 HC NH 2 HC NH 2

COOH C C
O O O
O

Cu

b. Xanthoprotein test

The purpose of this experiment is to test the presence of aromatic structure of amino
acid (benzene). The benzene nuclei present in the tyrosine, phenylalanine, and tryptophan
molecules will be nitrated by the addition of HNO3. The nitro compounds that are formed are
yellow and in an alkaline environment will be ionized freely and the color becomes older or
turns to orange.(Panji,2013)

 Egg
First, added 3 mL of egg solution into test tube, then added 1 mL HNO3 concentarted.
The function of adding HNO3 concentarted for nitration occur in core of benzene in molecule of
protein so, as to form white precipitate that change into yellow after heating. Then, heated it
until the colour change into yellow. The function of heating is to break the polipeptide bond to
small arrangement so it can speed up the reaction. After that cooled it and added ammonia.
The function of adding amonia to give base condition, this amonia caused the solution become
orange solution, it means that the solution can react with alkali.

First, added 3 mL of milk solution into test tube, then added 1 mL HNO3
concentarted. The function of adding HNO3 concentarted for nitration occur in core of benzene
in molecule of protein so, as to form white precipitate that change into yellow after heating.
Then, heated it until the colour change into yellow. The function of heating is to break the
polipeptide bond to small arrangement so it can speed up the reaction. After that cooled it and
added ammonia. The function of adding amonia to give base condition, this amonia caused the
solution become orange solution, it means that the solution can react with alkali.

This reaction as bellow :

3. Ninhydrin test

The purpose of this experiment is to identify the precence of free α-amino acid in
sample. Ninhydrin is reagent that can react with all α-amino acid form purple solution complex
compound. (Nuraeni,2011)

 Egg
First, entered 3 mL of egg solution into test tube. Then, set the pH of sample until 7 with
litmus paper. Then, added 10 drops of 0,2 % ninhydrin reagent formed colourless solution.
Then, heated it for 10 minutes formed purple solution.Tthe purple solution is frem because the
reaction between ninhydrin and amino acid, ninhydrin as stronger ozidizing agent at pH 4-8 and
in pH 7 ninhydrin can react with amino acids, ammonia and primary amino groups form purple
solution after heated. Heated to speed up the reaction. The positive test is formed purple
solution indicate that there are free α-amino acid in sample.

 Milk
First, entered 3 mL of milk solution into test tube. Then, set the pH of sample until 7 with
litmus paper. Then, added 10 drops of 0,2 % ninhydrin reagent formed colourless solution.
Then, heated it for 10 minutes formed purple solution.Tthe purple solution is frem because the
reaction between ninhydrin and amino acid, ninhydrin as stronger ozidizing agent at pH 4-8 and
in pH 7 ninhydrin can react with amino acids, ammonia and primary amino groups form purple
solution after heated. Heated to speed up the reaction. The positive test is formed purple
solution indicate that there are free α-amino acid in sample.

This reaction as below:

4. Millon test

The purpose of this experiment is to identify the presence of amino acid that is tyrosin.
the postive presence of tyrosin that in solution form red solution caused reaction between
mercury in millon reagent react with hydroxyphenil of tyrosin. (Panji,2013)

 Egg
First, entered 2 mL of egg solution into test tube. Added 1 mL of HgSO4 1% solution (millon
reagent) to hydrolyze the protein into tyrosin. Then, heated it to speed up the reaction formed
yellow solution. After that, added 1 drop of NaNO2 1%. The function of adding NaNO2 1% is to
reducing Hg to form red precipitate. Then, heated again to form red complex solution, but in
our experiment form brown precipitate on the top of test tube.

The mecanism reaction is that the nitartion reaction of HNO3 form H+ and NO2-, where Hg
oxidizing to Hg+ in HNO3 solution then form salt with hydroxyl group from tyrosin. Then, tyrosin
will have been nitrated, so tyrosin acccept the adding of N=O that can reversible form N-OH
(hydroxyphenyl).

 Milk
First, entered 2 mL of milk solution into test tube. Added 1 mL of HgSO4 1% solution (millon
reagent) to hydrolyze the protein into tyrosin. Then, heated it to speed up the reaction formed
yellow solution. After that, added 1 drop of NaNO2 1%. The function of adding NaNO2 1% is to
reducing Hg to form red precipitate. Then, heated again to form red complex solution, but in
our experiment form brown precipitate.

The mecanism reaction is that the nitartion reaction of HNO3 form H+ and NO2-, where Hg
oxidizing to Hg+ in HNO3 solution then form salt with hydroxyl group from tyrosin. Then, tyrosin
will have been nitrated, so tyrosin acccept the adding of N=O that can reversible form N-OH
(hydroxyphenyl).

This reaction as below :

HOOC

H2N CH
+ HNO2
H2C OH

Tyrosine

HOOC
HOOC

H2N CH
H2N CH

H2C OH
H2C O

N O N OH

Hg2+
HOOC

H2N CH
HOOC
H2C O O N
HC NH2
Hg

N O O CH2

5. Hydrolysis of Protein and Sulphur Identification

A rapid heating method of hydrolysis by the use of microwave oven has been
applied to amino acid analysis of proteins and peptides. This convenient method has
been compared with the conventional 6 N HCl hydrolysis at 110° for 24 h. The
advantages of this new method are its expedition and the accurate and comparable
results as compared to the tedious conventional technique. The method provides a
rapid processing of multiple samples within minutes instead of days and inexpensive
access to the important data of amino acid compositions of proteins by the commonly
used microwave oven. The necessary change in the design of hydrolysis vials and the
safety precautions accompanying this novel use of microwave acid‐digestion method
are also described.

Various species of organisms cannot synthesize or are not efficient in generating

all of the twenty amino acids needed to construct the proteins and enzymes essential
for their survival. To sustain growth and to maintain metabolic functions, these amino
acids must be provided from outside sources. This can be accomplished by the intake of
proteins. Humans are a good example of living organisms that ingest proteins as part of
their nutritional requirements. However, protein molecules are generally quite large,
and these large molecules cannot be transported across cell membranes for the same
reason that you cannot bring an elephant through the door into your dorm room. Some
organisms secrete proteolytic enzymes extracellular to break down the protein to its
component monomeric amino acid units by hydrolyzing peptide bonds at the end of the
polymer chain. A series of shorter polypeptides of different lengths are also formed if
the broken peptide bonds are not at the end of the polymer chain. Thus, depending on
the location of the attack, proteases can be further classified into exopeptidases (attack
on the terminal group) and endopeptidases (attack on internal linkages).

In the previous experiment, the hydrolysis of a protein was monitored with the
release of a dye that was bound to the protein. In this experiment, another more
accurate and generally accepted color method is introduced. In this method, an organic
compound called ninhydrin is reacted with the amino acids released during the
hydrolysis of the protein. The original unreacted ninhydrin is yellowish in color, but the
reacted product of ninhydrin has a deep purple-blue color. For example, the procedure
given at the end of this section yields an absorbance of 0.27 for 1.X10 ^-4M of glutamic
acid. Since ninhydrin does not react with the undegraded protein, one can measure the
amino acid concentration by following the development of the purple color by
 measuring the absorbance of the solution with a spectrophotometer. Because the color
intensity is a measure of the amino acid present, the color should intensify as more
protein is degraded to amino acid over time. The upper limit in color intensity is reached
when all the ninhydrin originally present in the solution has been consumed. Thus, the
amount of ninhydrin originally present in the reaction mixture determines the maximum
amino acid concentration that can be detected.

This experiment will also introduce students to the fundamentals of

quantitative assays. First, when a spectrophotometer is used to quantitatively measure
the absorbance of a colored solution, an absorbance spectrum should be obtained to
determine the best wavelength to use. Thereafter, the same wavelength is used for all
the subsequent determinations. Secondly, when there is a gradual development of color
due to the limited reaction rate of the color reaction, the time needed for the
completion of the reaction should be determined from the plot of absorbance versus
time. Obviously, this time is when the change in the absorbance as a function of time is
no longer significant. Thirdly, a calibration curve must be obtained by subjecting
standard solutions of known concentrations to the same procedure so that the
absorbance measurement can be correlated to actual physical units, in this case, the
amino acid concentration. Finally, one should always remember to compare his sample
to some reference, in this case, the reference being the protein mixture with no
protease added. To obtain a blank reading, add ninhydrin reagent to the protein
solution in the absence of protease and find the absorbance from the ninhydrin
reaction. Protein solution in the absence of protease may not always give absolutely
negative results due to other contaminants present in the solution, or the protein itself
may contribute to the development of purple color to a certain degree.

The reaction as below :

Pb(CH3COO)2 (aq) + 4 NaOH(aq) → Na2PbO2 (aq)

Na2PbO2(aq) + 2S2- (aq) + 2H2O(l) → PbS(s) +2 NaOH(aq) +2OH-(aq)

X. Conclusion

1.a. The form of precipitate on the tube, indicate that acetate acid can cause the denaturation
of protein

b. The heating process can cause the denaturation of protein. Indicated by the form of
precipitate on the bottom of the tube.

c. The adding of formaldehyde in the tube can cause the denaturation of protein, indicated by
the form of precipitate on each tube.

2. Protein have amfoter charateristic indicate that the sample can react with acid and base and
form pink solution.

3. a. Precipitate of protein dissolved when added with aquadest. Indicate that reaction between
protein and (NH4)2SO4 is reversible reaction

b. Reaction of protein with HNO3 is reversible reaction indicated by the formation of precipitate
when added saturated HNO3. Meanwhile reaction of HCl with protein is reversible reaction
indicated by the precipitation dissolved when added saturated HCl.

c. Reaction of protein with heavy metals (ZnSO4, CuSO4, FeSO4, PbSO4, HgSO4) are reversible
reaction, indicated by the precipitation dissolved when added saturated heavy metals (ZnSO4,
CuSO4, FeSO4, PbSO4, HgSO4)

4. a. There are peptide bond in the sample indicate that form purple solution
 b. There are aromatic structure of protein in sample indicate that the solution form orange
solution

c. There are free α-amino acidin sample indicate that the solution change to purple solution

d. There are no tyrosin in sample indicate that there no red complex solution formed.

5. The use of NaOH is for cutting the protein bond, and thecolor change become brown,
indicated that there Sulphur compound in the protein (milk).

XI. Discussion

4. d. Millon test

In our experiments, the result is brown precipitate but it should red complex solution
because reaction between mercury in millon reagent react with hydroxyphenil of tyrosin
form red complex. The eror can ocuur because the tools are not clean and eror from
human.
References

Aji B.K. dan F. Kurniawan. 2012. Pemanfaatan Serbuk Biji Salak (Salacca Zalacca)
sebagai Adsorben Cr(IV) dengan Metode Batch dan Kolom Jurnal Sains POMITS.
1 (1): 1-6

Bruice, Paula Yurkanis. 2011. Organic Chemistry 6th Edition. United State of America:
Pearson Education Inc

Carey, Francis A. 2008. Organic Chemistry 7th Edition. New York: Mc-Graw-Hill

Fessenden, Ralp J. 1982. Kimia Organik Edisi Ketiga. Jakarta: Erlangga

Hidajati, Nurul et al. 2017. Buku Petunjuk Praktikum Kimia Organik. Surabaya: FMIPA
Universitas Negeri Surabaya

Himedia Laboratories. No year. HiPer® Protein Estimation Teaching Kit (Qualitative)(e-

book).http://himedialabs.com/td/htbc004.pdf. Accessed on April 8 April 2018

Mc Murry, John E. 2012. Organic Chemistry. Eight Edition, International Edition.

China: Mary Finch

Milio, Frank R. 1995. Qualitative Testing for Amino Acids and Proteins.(online).
http://labopslton.wikispaces.com/file/view/qualitative+testing+for+amino+acids+%
26+proteins.pdf accessed on April 8 April 2018

Nuraeni,Rini. 2013. Laporan Uji Ninhidrin. www.slideshare.net accesed on 9 April 2018

at 20.30 p.m

Panji. 2013. Uji Xanthoprotein. www.edubio.info accesed on 9 April 2018 at 20.18 p.m

Poedjiadi, A. 2007. Dasar – Dasar Biokimia. Edisi Revisi. Jakarta: UI Press

Rosik, Ernalia. 2015. Laporan Praktikum Biokimia Pangan Protein I Uji Biuret.
www.slideshare.net accesed on 9 April 2018 at 19.07 p.m

Sinaga, Masdalena et. al. No year. Pemekatan Enzim Selulase Penicilium sp.
LBKURCC20 Dengan Pengendapan Amonium Sulfat 80% Jenuh.
Syabana, M.F. 2011. Sifat Asam Amino(online).
http://nurul.kimia.upi.edu/arsipkuliah/web2011/0800521/sifatasamamino.html.
Accessed on April 9 2018

Vina. 2015. Uji Protein (Uji Biuret) www.vinaoktap2015.wordpress.com accesed on 9

April 2018 at 19.47 p.m

Wade, L.G. 2013. Organic Chemistry 8th Edition. United State of America: Pearson
Education
XII. Attachment

1. Question’s Answer
2. Explain what is the function of testing the protein with each test reagent (CuSO4, HgCl2,
HNO3, Pb acetate)!
a. CuSO4 is used to test for the presence of heavy metals in proteins characterized by
presence of precipitation when positive proteins contain heavy metals.
b. HgCl2 is used for protein tests containing phenyl hydroxyl groups (-OH).
c. HNO3 was used to test for the presence of benzene rings from the amino acid salt of
the protein compound, ie in this experiment when concentrated nitric acid was added and
produced nitrobenzene derivatives.
d. Acetate pb is used to test for the presence of cysteine and methionine amino acids,
which in this experiment will produce a black color solution because the S atom reacts
with acetic acid to form PbS precipitate.

2. What is the effect of organic solvent (acetone, ethanol) on protein denaturation properties?

Pengaruh pelarut organic (aseton, etanol) terhadap sifat denaturasi protein adalah protein
atau asam nukleat akan kehilangan struktur sekunder dan tersiernya karena pelarut organic
mengakibatkan protein dapat terdenaturasi.

3. Mention the various bonds that cause the polypeptide to be stable in helical form!

a. Disulfide bond

Formed between 2 cysteine residues interconnected 2 parts of the polypeptide chain

through cysteine residues.

b. Hydrogen bond
 Formed between the groups of NH-
or -OH and the C = Odalam groups

No Pictures Explanation
.

of peptide bonds or -COO- in the R group

2. Documentation

1. Diluted the protein solution (egg)

with 5 mL egg and added water
up to 25 mL. This sample is usd to
all experiments.

2. The result of denaturation of

protein.

The result protein in acid and base

condition with milk as sample.
The resullt precipitation of protein
with ammonium sulphate

The result precipitation of protein

precipitation with mineral acid.
The sample is milk

The result precipitation of protein

precipitation with mineral acid.
The sample is egg.
Ditinguished the result of protein
precipitation with mineral acid,
with egg and milk as a sample
after heated.

The result protein precipitation

with heavy metal CuSO4

The result protein precipitation

with heavy metal ZnSO4
The result protein precipitation
with heavy metal HgSO4

The result protein precipitation

with heavy metal PbSO4

Distighusihed all protein

precipitation with
Cu,Hg,Zn,Pb,and Fe
The result of biuret test in protein.

The result of xantroprotein test in

protein.

The result of ninhydrin test in

protein.
The result of millon test in
protein.

The result of Hopcin-Cole

reaction with protein.

The result of hydrolisys and

sulphur identification.