Lapres Protein

I.
Title of Experiment : Understanding Characteristic and Color Reaction of

Protein
II. Starting Experiment : Tuesday / April, 30th 2018, 10.20 AM
Finishing Experiment : Tuesday / April, 03th 2018, 16.20 PM
III. The Aim of Experiment :
1. Distinguish the solubility of protein in a reversible and irreversible way.
2. Distinguish protein denaturation reaction caused acid, salt, salt from heavy metal, as
well as heated based on observation.
3. Understanding the causes of precipitate of protein.
4. Identify the presence of protein through a color reaction.
IV. Basic Theory
A. Amino Acid
Amino acids are carboxylic acids that contain an amine function. Under certain
conditions the amine group of one molecule and the carboxyl group of a second can
react, uniting the two amino acids by an amide bond.
Picture 1. Amino Acid

Carey, 2000.
Amino acids are classified as , ,  and so on, according to the location of the
amine group on the carbon chain that contains the carboxylic acid function.
Picture 2. The example from Classification of amino acid

Carey, 2000.
B. Acid Base Behavior of Amino Acid
The carboxyl group is deprotonated and exists as the carboxylate anion at a
physiological pH of 7.3, while an amino group is protonated and exists as the
Protein 1
ammonium cation. Thus, amino acids exist in aqueous solution primarily in the form
of a dipolar ion, or zwitterion (German zwitter, meaning “hybrid”).
Amino acid zwitterions are internal salts and therefore have many of the physical
properties associated with salts. They have large dipole moments, are soluble in water
but insoluble in hydrocarbons, and are crystalline substances with relatively high
melting points. In addition, amino acids are amphiprotic; they can react either as acids
or as bases, depending on the circumstances. In aqueous acid solution, an amino acid
zwitterion is a base that accepts a proton to yield a cation; in aqueous base solution,
the zwitterion is an acid that loses a proton to form an anion. Note that it is the
carboxylate, CO2, that acts as the basic site and accepts a proton in acid solution, and it
is the ammonium cation, NH3. That acts as the acidic site and donates a proton in base
solution McMurry, 2008.
Picture 3. Structure in acid and base solution

McMurry, 2008.
The dipolar nature of amino acid gives them some unusual properties
1. Amino acid have high melting points, generally over 200C.
2. Amino acid are more soluble in water than they are in ether,
dichloromethane, and other common organic solvent.
3. Amino acids have much larger dipole moment than simple amines or
simple acids.
4. Amino acid are less acidic than most carboxylic acid and less basic than
most amines. In fact, the acidic part of the amino acid molecule is the –
NH3 group not a –COOH group. The basic part is the –COO- group, and
not a free –NH2 group.
R – COOH R – NH2 H3N – CHR – COO- (Wade, 2013).
Protein 2
C. The standart of amino acid in Protein
The standard amino acids are 20 common -amino acids that are found in
nearly all proteins. The standard amino acids differ from each other in the structure of
the side chains bonded to their carbon atoms. All the standard amino acids are L-
amino acids (Wade, 2013).
Protein 3
Picture 4. Standart Amino Acid (Wade,2013).
D. Isoelectric Point
An amino acid bears a positive charge in acidic solution (low pH) and a
negative charge in basic solution (high pH). There must be an intermediate pH where
the amino acid is evenly balanced between the two forms, as the dipolar zwitterion
with a net charge of zero. This pH is called the isoelectric pH or the isoelectric point,
abbreviated pI (Wade, 2013).
Picture 5. Isoelectric Point (McMurry, 2008).

More specifically, the pI of any amino acid is the average of the two
aciddissociation constants that involve the neutral zwitterion. For the 13 amino acids
with a neutral side chain, pI is the average of pKa1 and pKa2. For the four amino acids
with either a strongly or weakly acidic side chain, pI is the average of the two lowest
pKa values. For the three amino acids with a basic side chain, pI is the average of the
two highest pKa values.
Protein 4
E. Reactions of Amino Acids
1. Esterification of the Carboxyl Group
Like monofunctional carboxylic acids, amino acids are esterified by
treatment with a large excess of an alcohol and an acidic catalyst (often gaseous
HCl). Under these acidic conditions, the amino group is present in its protonated
NH3+ form, so it does not interfere with esterification. The following example
illustrates esterification of an amino acid.
Picture 6. Esterification of an amino acid.

Esters of amino acids are often used as protected derivatives to prevent the
carboxyl group from reacting in some undesired manner. Methyl, ethyl, and
benzyl esters are the most common protecting groups. Aqueous acid hydrolyzes
the ester and regenerates the free amino acid. Benzyl esters are particularly useful
as protecting groups because they can be removed either by acidic hydrolysis or
by neutral hydrogenolysis (“breaking apart by addition of hydrogen”). Catalytic
hydrogenation cleaves the benzyl ester, converting the benzyl group to toluene
and leaving the deprotected amino acid. Although the mechanism of this
hydrogenolysis is not well known, it apparently hinges on the ease of formation of
benzylic intermediates (Wade, 2013).
2. Reaction with Ninhydrin

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. The side
chain of the amino acid is lost as an aldehyde (Wade, 2013).
Protein 5
Picture 7. Reaction with Nynhydrin
(Wade, 2013).
F. Peptide Structure
The most important reaction of amino acids is the formation of peptide bonds.
Amines and acids can condense, with the loss of water, to form amides. Industrial
processes often make amides simply by mixing the acid and the amine, then heating
the mixture to drive off water (Wade, 2013).
The amide nitrogen is no longer a strong base, and the C-N bond has restricted
rotation because of its partial double-bond character. In a peptide, this partial double
bond character results in six atoms being held rather rigidly in a plane. Having both an
amino group and a carboxyl group, an amino acid is ideally suited to form an amide
linkage. Under the proper conditions, the amino group of one molecule condenses
with the carboxyl group of another. The product is an amide called a dipeptide because
it consists of two amino acids. The amide linkage between the amino acids is called a
peptide bond. A peptide is a compound containing two or more amino acids linked
by amide bonds between the amino group of each amino acid and the carboxyl group
of the neighboring amino acid.
Picture 8. Peptide Bond

(Wadde, 2013 )
G. Protein
Proteins may be classified according to their chemical composition, their
shape, or their function. Protein composition and function are treated in detail in a
Protein 6
biochemistry course. For now, we briefly survey the types of proteins and their
general classifications. Proteins are grouped into simple and conjugated proteins
according to their chemical composition. Simple proteins are those that hydrolyze to
give only amino acids. All the protein structures we have considered so far are simple
proteins. Examples are insulin, ribonuclease, oxytocin, and bradykinin. Conjugated
proteins are bonded to a nonprotein prosthetic group such as a sugar, a nucleic acid, a
lipid, or some other group (Carey, 2000).
Table 1. Classes of Conjugated Proteins.

(Wade, 2013).
Proteins are classified as fibrous or globular depending on whether they form
long filaments or coil up on themselves. Fibrous proteins are stringy, tough, and
usually insoluble in water. They function primarily as structural parts of the organism.
Examples of fibrous proteins are in hooves and fingernails, and collagen in tendons.
Globular proteins are folded into roughly spherical shapes. They usually function as
enzymes, hormones, or transport proteins. Enzymes are protein-containing biological
catalysts; an example is ribonuclease, which cleaves RNA. Hormones help to regulate
processes in the body. An example is insulin, which regulates glucose levels in the
blood and its uptake by cells. Transport proteins bind to specific molecules and
transport them in the blood or through the cell membrane. An example is hemoglobin,
which transports oxygen in the blood from the lungs to the tissues (Wade, 2013).
1. Levels of Protein Structure
 The primary structure
The primary structure of a protein is simply the amino acid sequence
(McMurry,). This definition includes the sequence of amino acids, together with
any disulfide bridges. All the properties of the protein are determined, directly or
indirectly, by the primary structure. Any folding, hydrogen bonding, or catalytic
activity depends on the proper primary structure (Wade, 2013).
Protein 7
 The secondary structure
The secondary structure of a protein describes how segments of the peptide
(McMurry, ). In particular, the carbonyl oxygen atomsform hydrogen bonds with
the amide hydrogens. This tendency leads to orderly patterns of hydrogen bonding:
the helix and the pleated sheet. These hydrogen-bonded arrangements, if present,
are called the secondary structure of the protein (Wade, 2013).
 The tertiary structure
The tertiary structure of a protein is its complete three-dimensional
conformation. Think of the secondary structure as a spatial pattern in a local region
of the molecule. Parts of the protein may have the structure, while other parts may
have a-helical the pleated-sheet structure, and still other parts may be random
coils. The tertiary structure includes all the secondary structure and all the kinks
and folds in between (Wade, 2013).
 The Quarternary Structure
Quaternary structure refers to the association of two or more peptide
chains in the complete protein. Not all proteins have quaternary structure. The
ones that do are those that associate together in their active form. For example,
hemoglobin, the oxygen carrier in mammalian blood, consists of four peptide
chains fitted together to form a globular protein (McMurry, 2008).
2. Denaturation of Protein
For a protein to be biologically active, it must have the correct structure at all
levels. The sequence of amino acids must be right, with the correct disulfide bridges
linking the cysteines on the chains. The secondary and tertiary structures are
important, as well. The protein must be folded into its natural conformation, with the
appropriate areas of helix and pleated sheet. For an enzyme, the active site must have
the right conformation, with the necessary side-chain functional groups in the correct
positions. Conjugated proteins must have the right prosthetic groups, and multichain
proteins must have the right combination of individual peptides. With the exception of
the covalent primary structure, all these levels of structure are maintained by weak
solvation and hydrogen-bonding forces. Small changes in the environment can cause a
chemical or conformational change resulting in denaturation: disruption of the
normal structure and loss of biological activity. Many factors can cause denaturation,
but the most common ones are heat and pH (Wade, 2013)
a) Reversible and Irreversible Denaturation
Protein 8
When a protein is subjected to an acidic pH, some of the side-chain
carboxyl groups become protonated and lose their ionic charge. Conformational
changes result, leading to denaturation. In a basic solution, amino groups become
deprotonated, similarly losing their ionic charge, causing conformational changes
and denaturation. Milk turns sour because of the bacterial conversion of
carbohydrates to lactic acid. When the pH becomes strongly acidic, soluble
proteins in milk are denatured. and precipitate. This process is called curdling.
Some proteins are more resistant to acidic and basic conditions than others. For
example, most digestive enzymes such as amylase and trypsin remain active under
acidic conditions in the stomach, even at a pH of about 1. In many cases,
denaturation is irreversible. When cooked egg white is cooled, it does not become
uncooked. Curdled milk does not uncurdle when it is neutralized (Wade, 2013).
Denaturation may be reversible, however, if the protein has undergone only
mild denaturing conditions. For example, a protein can be salted out of solution by
a high salt concentration, which denatures and precipitates the protein. When the
precipitated protein is redissolved in a solution with a lower salt concentration, it
usually regains its activity together with its natural conformation (Wade, 2013 ).
b) Denaturation cuses heat
Heat can be used to disrupt hydrogen bonding and non-polar hydrophobic
interactions. This happens because high temperatures can increase the kinetic
energy and cause protein molecules to move or vibrate very quickly, thus
disrupting the bonds of these molecules. The egg protein is denatured and
coagulated during cooking. Some foods are cooked to denature the protein
contained in order to facilitate digestive enzymes in digesting the protein (Ophart,
C.E., 2003).
The heating will make the denatured protein so the ability to bind the water
decreases. This happens because heat energy will lead to disconnection of non-
covalent interactions that exist in the natural structure of the protein but does not
break its covalent bond in the form of peptide bonds. This process usually takes
place at a narrow temperature range (Ophart, C.E., 2003).
c) Denaturation causes acids and bases
The protein will experience the biggest turbidity when it reaches the
isoelectric pH, where the protein has the same positive and negative charge, at
which time the protein undergoes denaturation marked by increased turbidity and
Protein 9
incidence of clots. (Anna, P, 1994). Acids and bases can disrupt salt bridges in the
presence of ionic charges. A double-replacement reaction type occurs when
positive and negative ions in the salt alternate with positive and negative ions from
the added acid or base. This reaction occurs in the digestive system, when gastric
acid coagulates the milk consumed (Ophart, C.E., 2003).
d) Denaturation cause heavy metal
Heavy metal salts denature the same protein as acid and base. Heavy metal
salts generally contain Hg2 +, Pb2 +, Ag + Ti +, Cd2 + and other metals with large
atomic weights. The reaction that occurs between heavy metal salts will result in
the formation of insoluble metal-protein salts (Ophart, C.E., 2003).
Proteins will experience precipitation when reacting with metal ions.
Precipitation by positive ions (metal) is required ph above solution pi because the
protein is negatively charged, precipitation by negative ions is required ph solution
below pi because the protein is positively charged. Positive ions that can
precipitate proteins are; Ag+, Ca2+, Zn2+, Hg2+, Fe2+, Cu2+ and Pb2+, whereas
negative ions that precipitate proteins are; salicylate ions, trichloracetate,
pichtrates, tannins and sulfosalisilate. (Anna, P., 1994).
e) Denaturation cause the addition of chemical compounds
Can be caused by chemical reaction between the existing groups with the
added compound. For example, the addition of formaldehyde will react to the
amino group in the protein with the aminodimethyl acid. The result of this reaction
gives a precipitate that is not soluble in water and hardened. (Anwar, et al. 1994).
3. Precipitation of Protein
The presence of various functional groups (NH2, NH, OH, CO) and the form of
double ions (zwitter ion) contained in the protein structure can lead to the reaction of
protein precipitation. The functional groups are capable of binding water molecules
through the formation of hydrogen bonds. Deposition reaction may occur due to the
addition of chemicals such as salts and organic solvents which can change the
solubility of proteins in water (Hidajati, et al. 2017).
a) Precipitation with ammonium sulfate
The precipitation caused by the addition of concentrated ammonium sulfate
causes the dehydration of proteins (water loss). As a result of this dehydratation
process, protein molecules that have the smallest solubility will easily settle.
Protein deposited in this way does not undergo chemical changes so that it can be
Protein 10
easily reconstituted by adding water. Deposition in this way is reversible (Hidajati,
et al. 2017).
b) Precipitation due to concentrated mineral acids
Treatment of concentrated mineral acids in proteins can lead to the
formation of salt compounds from acidic reactions with amino groups of proteins.
Another effect can be irreversible denaturation and protein precipitation is
obtained. However, in general, deposition with the addition of strong mineral acids
(except concentrated HNO3) is reversible (Hidajati, et al. 2017).
c) Precipitation of protein by heavy metals
The basis of the precipitation reaction by heavy metals is the neutralization
of the charge. Precipitation may occur when the protein is in a negatively charged
isoelectric form. Given the positive charge of heavy metals there will be a
neutralization reaction of the protein and a salt of precipitated proteinic salt. The
precipitate of this protein will dissolve again in the addition of alkali (eg NH3 and
NaOH). The precipitation properties of these proteins are reversible (Hidajati, et
al. 2017).
4. Color Reaction of Protein
a) Biuret Reaction
Biuret reactions are common color reactions used for peptide (-CO-NH-)
and protein groups. The positive reaction is characterized by the formation of
purple color due to the formation of complex compounds between Cu2 + and N of
peptide bond molecules. The amount of amino acid attached to the peptide bond
affects the color of this reaction. The compound with dipeptide provides blue,
purple tripeptide and tetrapeptide as well as a complex peptide giving it a red
color. Some proteins that have clusters -CS-NH, -CH-NH- in their molecules also
provide a positive color test of this biuretic reaction forming a complex compound
illustrated below:
Protein 11
Picture 9. Complex stucture of Cu2+ peptide compound
(Hidajati, et al. 2017).
b) Ksanthoprotein reaction
Ksanthoprotein color reactions can occur due to the nitration reaction of the
benzene ring of the protein amino acids. The test is said to be positively indicated
by the yellow color caused by the formation of a polynitrobenzene compound of
the amino acid protein. This reaction is positive for proteins containing amino
acids with benzene nuclei such as tyrosine, phenyl alamin, tryptophan. In addition
the yellow-colored alkali compounds will disappear and turn yellow to orange due
to the acidity of the phenol reacting with the alkali. This orange color when
acidified will turn back to yellow (Hidajati, et al. 2017).
c) Ninhydrin Reaction
The color reaction of the ninhydrin protein is positive when it gives color
blue or purple. This reaction occurs in the amino-free group of amino acids with
the ninhydrin listed below: The above-mentioned blue-violet color can also be
used to determine amino acids quantitatively by measuring their absorbance at 570
nm. The basis of this reaction is used in the tool for the determination of amino
acids (Hidajati, et al. 2017).
Protein 12
Picture 10. Ninhydrin Reaction
d) Millon's reaction
Millon reagents involve the addition of Hg compounds into proteins so that
the addition of these metals will produce white deposits of mercury compounds.
For proteins containing tyrosine or tryptophan the addition of Millon reagents
results in a red color. However, this reagent is not specific because it also gives a
positive test of red color in the presence of phenol compounds (Hidajati, et al.
2017).
e) Hopkin-Cole reaction
The color reaction of these proteins shows positive when marked the
formation of purple rings on the boundary plane between protein and reagent
solution. The formation of this ring is due to the formation of condensation of 2
indol nuclei from tryptophan with aldehyde. The disinidated aldehyde is obtained
by glycosalic acid used for the Adamkiewicz Hopkins test (Hidajati, et al. 2017).
5. Hydrolisis of Protein
The addition of alkali in proteins can lead to the hydrolysis of peptide bonds of the
protein polymer. This hydrolysis produces amino acid monomers and some amino
groups that turn ammonia. Due to the hydrolysis the amount of amino groups
decreases. If in proteins there are amino acids that have S atoms such as cysteine and
cystine in their molecules then these amino acids can be eliminated into the form of
H2S compounds. the addition of Pb salt in an alkaline atmosphere to an easily
observed PbS precipitate.
Pb2+ + 4OH- → PbO22- + 2 H2O
S2- + 2 H2O + PbO22- → PbS ↓ + 4OH- (Hidajati, et al. 2017).
Protein 13
V. Tools and Material
Tool:
 Test tube 20 pieces

 Beaker glass 50 mL 2 pieces
 Beaker glass 100 mL 2 pieces
 Beaker glass 250 mL 1 piece
 Bunsen burner 1 piece
 Tripod 1 piece
Materials :
 Egg enough
 Milk enough
 Acetate acid 10 mL
 (NH4)2SO4 solution 5 mL
 NaOH solution 50 mL
 HNO3 solution 5 mL
 CuSO4 solution 20 mL
 PbSO4 solution 20 mL
 HgSO4 solution 5 mL
 NaNO2 solution 5 mL
 Ammonium sulphate solution 5 mL
 Nynhidrin solution 10 mL
 Formaldehyde solution 5 mL
 Mercurysulphate solution 5 mL
 Pb acetate 5 mL
 HNO3 solution 5 mL
 Kongo indicator enough
 PP indicator enough
Protein 14
VI. Lanes Work
1. Denaturation of Protein
a. Denaturation because of acetate addition
5 mL protein solution
- Entered into test tube

- Added 2 drops acetate acid 1N
- 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 of protein solution

- Heated until 1 minute (the precipitate formed)
- Cooled
- Divided it into two test tube
Test tube 1 Test tube 2

- Added 1-2 drops of (NH4)2SO4 - Heated
- Heated it it
Result Result
c. Denaturation because of Formaldehyde

1-1,5 ml formaldehyde 2 ml aquadest

- Added protein solution
drop by drop
- Observed the precipitate
Result
Protein 15
2. Amfoter Characteristic of Protein
a. Test in acid condition
3 mL of aquadest
- Added 1 drop HCl 1N
- Added several drops congo indicator
- Added 2-3 mL of protein solution
- Noted the color changing
Result
b. Test in base condition
3 mL of NaOH 0,1 M 2-3 ml protein solution
- Entered into test tube. - Entered into test tube

- Added several drops of - Added drop by drop NaOH 0,1 M
PP indicator - Observed the color formed
Result
Result
a. Precipitation of protein with ammonium sulphate
3-4 mL of protein solution
- 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 HNO
3

- Tilted the tube
- Added 1-1,5 mL protein solution drop by drop through tube wall
- Let it stand until the white ring formed as protein precipitate
- Shaken it
- Added HNO3 again. Until formed white precipitate
Result
Protein 16
1-2 mL of concentrated HCl

- Tilted the tube
- Added 1-1,5 mL protein solution
- Taken up the tube again
- Let it stand until the 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 CuSO4 drop by drop - Added PbSO4 drop by drop
- Shaken
Blue it
precipitate - Shaken
White it
precipitate
- Added CuSO4 drop by drop, shaken - Added PbSO4 drop by drop,
it until precipitate dilute shaken it until precipitate dilute

- Repeated again with salt from Zn, - Repeated again with salt from Zn,
Fe, and Hg Fe, and Hg
- Observed the changing process - Observed the changing process
Result Result
4. Reaction the Color of Protein

a. Biuret reaction
3 ml Protein
- Added 1 ml 40% NaOH
- Added drop by drop 0,5% CuSO4, the color
become red or purple
Result
Protein 17
b. Xanthoprotein reaction
3 ml Protein solution

- Added 1 ml of concentrated HNO3
- Heated until formed yellow solution Cooled it
- Added amonia until formed orange solution
c. Ninhydrin reaction
Result
Protein solution 0.5%
- Set its pH to 7
- Taken 1 ml of this solution
- Added 10 drops of nynhydrin 0.2% solution
- Heated on 100˚C for 10 minutes
- Observed the changes
Result
d. Millon reaction
2 ml of protein
solution
- Added 1 ml mercury reagent (1% HgSO4 diluted in 10% H2SO4)
- Heated it, formed yellow solution
- Cooled it
- Added 1 drop of 1 % NaNO2 solution
- Heated it again. Formed red solution
Result
e. Hopkine-Cole Reaction
1 ml of
protein
- Added 1 drop of formaldehyde solution
- Added 1 drop of mercurysulphate reagent
- Added 1 ml concentrated sulfuric acid through the wall of
test tube until it formed two layers. There are purple ring,
if it shaken all of the solution become purple
Result
Protein 18
5. Protein Hydrolysis and Sulphur Identification
1 ml protein solution

- Added 1 ml of NaOH 40%
- Heated for 1 minute
- Added one drop of Pb-acetic formed black solution (indicate Pb)
Result
Protein 19
VII. Observation Result
Result of the Experiment
No. Experiment Procedure Assumpsion / Reaction Conclusion
Before After
1. Denaturation of Protein Egg : turbid Egg + Protein solution if reacted Protein in egg and milk is
a. Denaturation because of acetate addition solution CH3COOH : with acetate acid causing denaturated causes
5 mL protein turbid + white denaturation and formed adding acetic acid, it is

solution Milk : white precipitate precipitate marked white precipitate.
- Added 2 drops acetate acid 1N solution
- Shaken it. Flake will be formed Heated : turbid
- Heated in steam bath for 5 minutes
+ white
- Observed the precipitate changes
precipitate
Result
Milk +
CH3COOH :
white solution
+ precipitate
Protein 20
b. Denaturation because of heating Egg : turbid Egg heated Protein solution will causing Protein in egg and milk is
2-3 mL of protein solution solution until 1 minute : denaturation in high denaturated causes
- Entered into test tube turbid and temperature. Adding heating process. It is
- Heated until 1 minute (the precipitate
formed) Milk : white white (NH4)2SO4 as protein marked white precipitate.
- Cooled solution precipitate dehydrator, so protein lost
- Divided it into two test tube
H2O
Milk heated
Test tube Test tube until 1 minute :
1 - Added 1-2 drops of 2
white solution
(NH4)2SO4 - Heated it
- Heated it
EGG (heated)
Result Result
Tube I +
(NH4)2SO4 :
turbid solution
Protein 21
Tube II : turbid
solution
MILK :
Tube I +
(NH4)2SO4 :
white solution
Tube II :
white solution
c. Denaturation because of Formaldehyde Formaldehyd Formaldehyde Adding formaldehyde is Protein in egg and milk is
e : colorless + aquadest : causing amino group in denaturated caused
1-15 ml 2 ml aquadest
solution colorless protein reagent, yield amino formaldehyde compound.
formaldehyd
solution acid dimethyl (formed It is marked with
- Entered into test tube Aquadest : protein precipitate) and precipitate.

- Added protein solution colorless +egg (10 protein will be denaturation
drop by drop
- Observed the solution drops) : turbid
+ white
Result
precipitate
+milk (27
drops) white +
Protein 22
white
precipitate
2. Amfoter Characteristic of Protein Aquadest : Aquadest + Protein in egg and milk is

a. Test in acid condition colorless HCl : colorless (aq) + amino acid compound
solution solution H+(aq) ⇄ that have characteristic
3 mL of aquadest
amphoter compound
- Entered into test tube HCl : + cango because it is reacted with
- Added 1 drop HCl 1N (aq)
- Added several drops congo indicator colorless indicator (5 acid and base, it is
- Added 2-3 mL of protein solution solution drops) : purple maeked with white
- Noted the color changing Acid condition
solution (++) precipitate that form
When the acid is added, the
Result Cango
properties of the protein act
indicator : + egg : pink
as a base. This is because
orange solution with
proteins have amphoteric
solution white
properties (Anwar, C et al:
precipitate
1996).
+ milk : pink
solution with
white
precipitate
Protein 23
b. Test in base condition NaOH : NaOH + PP (1
colorless drop) : pink (aq) +
3 mL of NaOH 0,1 M
solution solution OH-(aq) ⇄
- Entered into test tube.
- Added several drops of
PP indicator PP : colorless Egg + NaOH : (aq) +
solution colorless H2O(aq)
Result
solution Bases condition
When added bases, the
2-3 ml protein solution Milk + NaOH : properties of proteins are
white solution acidic. This is because
- Added drop by drop NaOH proteins have amphoteric
0,1 M
Egg + NaOH + properties (Anwar, C et al:
- Observed the color formed
PP : pink 1996).
Result
(+++) solution
Milk + NaOH
+ PP: pink
(++) solution
Protein 24
3. Precipitation of Protein Egg : EGG : (NH4)2SO4(aq) + nH2O(l) Protein in egg and milk
a. Precipitation of protein with ammonium colorless +ammonium can precipitating when
 (NH4)2SO4.nH2(aq)
sulphate soluion sulphate : added with ammonium
turbid solution sulphate. Then when
3-4 mL of protein
Milk : white added by aquadest the
- Added 3-4 mL saturated ammonium solution + aquadest + protein can diluted that is
sulphate shake : showing it the
- Shaken it slowly (the solution
Ammonium colorless, little experiment is reversible.
become turbid)
- Added 2-3 mL aquadest sulphate : flake
- Shaken it again. colorless
Result
MILK :
Aquadest : +ammonium
colorless sulphate :
solution white solution
HNO3 : + aquadest +
colorless shake :; on
solution bottom
colorless, on
top white
solution
Protein 25
b. Protein precipitation with mineral acid HNO3 : HNO3 + egg : Test with concentrated Protein of egg and milk
colorless formed ring HNO3 can formed ring (white)
1 mL of HNO3
when added by HNO3,
- Poured into test tube HCl : when shaken it and
- Tilted the tube + NO3-
colorless + HNO3 : added HNO3 again, this
- Added 1-1,5 mL protein solution
drop by drop through tube wall yellow precipitate can diluted.
- Let it stand until the white ring precipitate,  NO3- + This experiment is
formed as protein precipitate
- Shaken it bottom irreversible because
- Added HNO3 again. Until formed colorless Test with concentrated HCl remain of precipitate
white precipitate containing of in tube.
HNO3 + milk :
Result + Cl- 
formed ring
1-2 mL of concentrated HCl
+HNO3 : Cl- +
- Tilted the tube yellow
- Added 1-1,5 mL protein solution precipitate and
- Taken up the tube again
- Let it stand until the white ring the bottom
formed as protein precipitate colorless
- Shaken it
- Added HCl again. Formed clear
solution HCl + egg :
formed white
Result
Protein 26
ring
+ HCl :
colorless (30
drops)
HCl + milk :
formed white
ring
+ HCl :
colorless (±200
drops)
Protein 27
c. Protein precipitation with heavy metal CuSO4 : blue Egg + CuSO4 : With CuSO4 Protein of egg and milk
solution blue can forming precipitate if
1-1,5 mL Protein solution precipitate heated some heavy metal
+ Cu2+ ⇌
- Poured into test tube PbSO4 : (Cu, Pb, Zn, Fe, and Hg).
- Added CuSO4 drop by drop
colorless + CuSO4 (5 Then if added again with
- Shaken
Blue it
precipitate
solution drops) : the some heavy metal,
- Added CuSO4 drop by drop,
diluted, blue then it diluted. That is
shaken it until precipitate dilute
ZnSO4 : solution showing if this
- Repeated again with salt from With PbSO4
Zn, Fe, and Hg colorless experiment is reversible.
- Observed the changing process solution Milk + CuSO4
Result : blue + Pb2+ ⇌
FeSO4 : little precipitate
orange
solution + CuSO4 (20
drops) : With ZnSO4
HgSO4 : diluted, blue
colorless solution +Zn2+ ⇌
soluttion
Egg + PbSO4 :
colorless
precipitate With FeSO4
Protein 28
+ PbSO4 (3
+Fe2+ ⇌
drops) :
colorless
solution
With HgSO4
Milk + PbSO4
: white
+Hg2+ ⇌
precipitate
+ PbSO4 (3
drops) : white
solution
Egg + ZnSO4 :
white
precipitate
+ ZnSO4 (3
drops) : diluted
solution
Protein 29
Milk + ZnSO4
: white
precipitate
+ZnSO4 (3
drops) : diluted
solution
Protein 30
4. Reaction the Color of Protein Egg : Egg solution + CuSO4 (aq) + 2 NaOH (aq) Protein in egg solution
a. Biuret reaction colorless, NaOH solution → Cu(OH)2 (aq) + Na2SO4 and milk can formed
little turbid : colorless (aq) purple solution if reacted
3 ml solution Cu(OH)2 (aq) → Cu2+ with biuret reaction
- Entered into test tube Milk : white +2OH-
- Added 1 ml 40% NaOH
solution Milk + NaOH :
- Added drop by drop 0,5% CuSO4, the
color become red or purple white solution
Cu2+
NaOH 40% :
Result colorless + CuSO4 (8
drops): purple,
CuSO4 0.5%: turbid
colorless
b. Xanthoprotein reaction HNO3 Egg + HNO3 : Protein in egg and milk

concentrated light yellowish contain amino acid wih
3 ml
: colorless solution benzene group that
Protein
- Entered into test tube + HNO3
indicate with orange
- Added 1 ml of concentrated HNO3
+ heated : solution in xanthoprotein
- Heated until formed yellow
solution Cooled it yellow and reaction
- Added amonia until formed orange white solution
solution
Result
Protein 31
+ ammonia :
orange
solution
Milk + HNO3 :
light yellowish
solution
+ heated :
yellow and
white solution,
turbid
+ ammonia :
orange
solution, turbid
Protein 32
c. Ninhydrin reaction Ninhydrin Egg + Protein in egg and milk
0.2 % : ninhydrin 0.2 contain α-amino acid that
Protein solution 0.5%
colorless % : colorless indicate from the color
- Set its pH to 7 solution blue purpleish with
- Taken 1 ml of this solution +
Millon ninhydrin reaction.
- Added 10 drops of nynhydrin 0.2%
solution reagent : + heated : blue
- Heated on 100˚C for 10 minutes colorless purpleish (++)
- Observed the changes 
solution
Result Milk +
NaNO2 1 % : ninhydrin 0.2
colorless % : turbid,
white solution
+ heated : blue + + CO2 + H2O

purpleish Possitive reaction marked
by formed Ruhemann
purple that indicate free
alpha amino acid
(Fessenden, 1986).
Protein 33
d. Millon reaction H2SO4 : Egg + millon Protein in egg and milk
2 ml of protein solution colorless reagent : white contain amino acid
- Entered into test tube solution turbid and + Hg2+ 2H+ + 2NO2-  (tyrocin and triptofon)
- Added 1 ml mercury reagent (1%
colorless
HgSO4 diluted in 10% H2SO4)
HgSO4 : solution
- Heated it, formed yellow solution
- Cooled it colorless
- Added 1 drop of 1 % NaNO2 solution + heated : red
solution precipitate + 2H2O
- Heated it again. Formed red solution
NaNO2 1 % : Millon test identyfy if
Result colorless + NaNO2 : red theprotein contain tyrosin
solution and that indicated by red
have flake complex (Poedjiadi, 2007).
Milk + millon
reagent : white
turbid solution
+ heated : red
precipitate
+ NaNO2 : red
solution and
red precipitate
Protein 34
e. Hopkine-Cole Reaction Formaldehyd Egg + Protein in milk and egg
1 ml of protein solution e : colorless formaldehyde : not contain indol groups
- Entered into test tube solution white solution, because the color not
- Added 1 drop of formaldehyde turbid became purple ring.
solution + 
- Added 1 drop of mercurysulphate Millon
reagent reagent : + millon :
- Added 1 ml concentrated sulfuric colorless white solution,
acid through the wall of test tube
solution turbid
until it formed two layers. There are
purple ring, if it shaken all of the
solution become purple + H2SO4
Possitive reaction marked
Result concentrated :
with form ring with the
grey solution
color is purple, its indicate if
the protein contain indol
Milk +
groups.
formaldehyde :
white solution,
turbid
+ millon :
white solution,
turbid
Protein 35
+ H2SO4
concentrated :
grey (++)
solution in the
bottom tube
and white
precipitate in
the top of tube
5. Protein Hydrolysis and Sulphur Identification Egg : Egg + NaOH Pb2+(aq) + 4OH-(aq) → Amino acid in egg
colorless 40%: colorless PbO22-(aq) + H2O(l) protein have more atom
1 ml protein little turbid solution S, because react with Pb-

solution S2-(aq) + 2H2O(aq) + PbO22- acetic.
- Added 1 ml of NaOH 40% Milk : white + heated : (aq) → PbS↓(s) + 4OH-(aq)
- Heated for 1 minute solution yellowish Amino acid in milk
- Added one drop of Pb-acetic formed solution protein haven’t more
Protein that contain amino
black solution (indicate Pb)
NaOH 40% : acid with atom s will give atom S, because the color
Result colorless +Pb-acetic : became green brownish
the color is black if react
solution brown blackish with Pb acetic if react with Pb-acetic.
solution
Pb-acetic :
Protein 36
colorless Milk + NaOH
solution 40%: white
solution, turbid
+ heated :
yellow, turbid
solution
+Pb-acetic :
green
brownish
Protein 37
VIII. Analysis and Discussion
The experiment which have do on Tuesday, April 3rd, 2018 with the title is
“Understanding Characteristic and Color Reaction” and have purpose there are : 1) to
differentiate proteins solubility’s property with reverrsible and irreverrsible, 2) to
differentiate denaturation reaction of proteins that caused by acid, salt, salt of heavy
metal, and heating based on observation, 3) to understand cause happend of
precipitated by proteins, and 4) to identify existance of proteins through color reaction.
This experiment use egg and milk as sources of protein. Egg are a source of
food that many people use. These food contain proteins, fats, vitamins, and minerals.
Meanwhile, milk is a solution that contains proteins. Lactose, certain minerals and
vitamins that emulsify fats and casein. Casein can be precipitated by acidifying milk to
pH 4,7, for non precipitated protein at pH 4,7 it is present serum (Anwar, et al, 1996).
1. Denaturation of Protein.
For first experiment, test about denaturation of proteins. In this experiment,
consist of three method to denaturazed proteins, there are acetate addition, by heat,
and formaldehyde.
First method is acetate addition.
Prepare proteins solution, for egg, before used it crack the eggshell then
separated between yolk and white. That use for experiment is white egg, take about 5
ml of white egg. After that, the white egg diluted with aquadest until 25 ml. Diluted
have purpose to make protein solution, because if we use white egg in direct that’s
mean not solution. Because texture of white egg not solution. For milk, can use in
direct, not diluted before.
Arabic chicken’s egg
From 25 ml protein solution of egg, take 5 ml of the solution and entered into
test tube, the color of egg solution is colorless with little flake. added by 2 drops of
CH3COOH 1 N. The purpose this added is to give acid condition, with it can causes
form of protein salt that undissolve. After added CH3COOH, the tube shaken, and
result flake. Then the tube heated in steam bath for 5 minutes. This heat have purpose
to test the mixture will be formed white precipitate or diluted and become colorless.
After waiting, truthly the mixture forming white precipitate, that is showing if the
protein denaturazed.
Characteristic of protein which denaturazed can be look from some even. One
of them is the changes of this physical structure. Proteins which have denaturation
Protein 38
usually occuring less solubility. Molecule layer of hydrophobic part will occur
position change from inside to outside, this conditions is continues. Like this protein
solution that we test have denaturazed caused by added of acetate acid.
Denaturation cause added by acid because zwitter ions in proteins. Proteins
can forming zwitter ions structure. Proteins also have isoelectric point, count of
positive charge and negative charge is equilibrium. So, that is condition make proteins
can denaturazed with forming precipitate and become turbid solution. The mechanism
is added by acetate acid will broke salt bridge in proteins.
Milk
Take 5 ml of milk and entered into test tube the color of milk is white. The
tube added by 2 drops of CH3COOH 1 N. The purpose this added is to give acid
condition, with it can causes form of protein salt that undissolve. After added
CH3COOH, the tube shaken, and result flake. Then the mixture heated in steam bath
for 5 minutes. This heat have purpose to test the mixture will be formed white
precipitate or diluted and become colorless. After waiting, truthly the mixture After
waiting, truthly the mixture forming white precipitate, that is showing if the protein
denaturazed.
Characteristic of protein which denaturazed can be look from some even. One
of them is the changes of this physical structure. Proteins which have denaturation
usually occuring less solubility. Molecule layer of hydrophobic part will occur
position change from inside to outside, this conditions is continues. Like this protein
solution that we test have denaturazed caused by added of acetate acid.
Denaturation cause added by acid because zwitter ions in proteins. Proteins
can forming zwitter ions structure. Proteins also have isoelectric point, count of
positive charge and negative charge is equilibrium. So, that is condition make proteins
can denaturazed with forming precipitate and become turbid solution. The mechanism
is added by acetate acid will broke salt bridge in proteins.
The second method is denaturation because heating.
 Arabic chicken’s egg
Take 2 -3 ml of proteins solution from solution which have made up. Egg
solution that colorless entered into test tube. Heated until 1 minutes, after heated,
egg become turbid and little white flake, then cooled. Next, the tube divided into
two test tube. Then give lable for each tube. For test tube 1 of egg solution, added
1 -2 drops of (NH4)2SO4 then heated. The function of (NH4)2SO4 is to give acid
Protein 39
and base condition in solution. Added of acid or base can broke salt bridge in
proteins which one of interaction in proteins, if will be broke then denaturation
will occur.
While, for test tube 2, just heated, not added anything. After each heated,
compare the result. So, the result of this experiment is egg become turbid, and
more white precipitate on added of (NH4)2SO4 and heating.
Denaturation can caused by heated. Heat can broke hydrogen bond in
proteins but not disturb its covalent bond. That is caused by increasing temperature
will make molecule kinethic energy increas. Beceause this increas will broke
hydrogen bond and make entalpi system changes be high. If proteins denaturazed,
that is showing if the entrophy increasing too. If highest entrophy occur, then
occur denaturation and disorder. That is make proteins for bonding water is
decrease and occur koagulation
 Milk
Take 2 -3 ml of milk then entered into test tube,the color of milk is white.
Heated until 1 minutes, after heated, milk white solution, cooled. Next, each tube
divided into two test tube. Then give lable for each tube. For test tube 1 of milk,
added 1 -2 drops of (NH4)2SO4 then heated. The function of (NH4)2SO4 is give
acid and base condition in solution. Added of acid or base can broke salt bridge in
proteins which one of interaction in proteins, if will be broke then denaturation
will occur.
While, for test tube 2, just heated, not added anything. After each heated,
compare the result. So, the result of this experiment is milk become white solution,
because the precipitate dilute (just heating) and white precipitate on added of
(NH4)2SO4 and heating.
Denaturation can caused by heated. Heat can broke hydrogen bond in
proteins but not disturb its covalent bond. That is caused by increasing temperature
will make molecule kinethic energy increas. Beceause this increas will broke
hydrogen bond and make entalpi system changes be high. If proteins denaturazed,
that is showing if the entrophy increasing too. If highest entrophy occur, then
occur denaturation and disorder. That is make proteins for bonding water is
decrease and occur koagulation
Protein 40
For last method is with formaldehyde.
 Arabic Chicken’s egg
1 – 1,5 ml of formaldehyde mix with 2 ml aquadest. Fromaldehyde which
mixed by aquadest become colorless solution. Then entered into test tube, after
that the tube added by protein solution drop by drop. Then observed the precipitate
which occur. For egg, resulting turbid solution and little flake. That is showing if
occur denaturation of proteins.
The flake which formed is reaction result of amino groups in proteins and
aminodimethyl acid. Amino acid that bonding with formaldehyde will reacted by
acid, beacuse formaldehyde bonded by amino group forming aminodimethyl acid
derivate which causes formed precipitate.
 Milk
Prepare 1 – 1,5 ml of formaldehyde mix with 2 ml aquadest.
Fromaldehyde which mixed by aquadest become colorless solution. Then entered
into test tube, after that the tube added by protein solution drop by drop. Then
observed the precipitate which occur. For milk, resulting white solution and little
white precipitate. That is showing if occur denaturation of proteins.
The flake which formed is reaction result of amino groups in proteins and
aminodimethyl acid. Amino acid that bonding with formaldehyde will reacted by
acid, beacuse formaldehyde bonded by amino group forming aminodimethyl acid
derivate which causes formed precipitate.
2. Amphoter Characteristic of Protein
For second experiment is about amphoter characteristic of Proteins. In this
experiment, separated become two experiment. That is in acid condition and abse
condition.
Acid condition
Arabic Chicken’s Egg
First, take 3 ml of aquadest. Then entered into test tube. After that, added 1
drop of colorless HCl 1 N and added several drops of Congo indicatore which red
color. The result of mixture is soft blue solution. Added 2 ml of egg solution and it
changes the color become soft pink solution. This color changes is showing if protein
have amphoter characteristic.
Amino acid contains of carboxylate ions (CO2-) and ammonium ion (NH3+) in
the same molecule corner. That is make proteins have amphoter characteristic, can
Protein 41
react with acid or base. Added of HCl have the purpose is give acid condition and
when dropped by Congo indicatore, the solution changes become soft pink solution,
that is show acid characteristic and amphoter of proteins.
In certain condition, amino group and carboxylate will netralizing, so the
molecule nothing charge, can be called isoelectric. If pH under isoelectric condition,
then the charge will be positive charge. Meanwhile, if pH upper isoelectric condition,
then the charge will be negative charge or netral.
Milk
First, take 3 ml of aquadest. Then entered into test tube. After that, added 1
drop of colorless HCl 1 N and added several drops of Congo indicatore which red
color. The result of mixture is soft blue solution. Added 2 ml of milk and it changes
the color become soft pink solution (+). This color changes is showing if protein have
amphoter characteristic.
Amino acid contains of carboxylate ions (CO2-) and ammonium ion (NH3+) in
the same molecule corner. That is make proteins have amphoter characteristic, can
react with acid or base. Added of HCl have the purpose is give acid condition and
when dropped by Congo indicatore, the solution changes become soft pink solution,
that is show acid characteristic and amphoter of proteins.
In certain condition, amino group and carboxylate will netralizing, so the
molecule nothing charge, can be called isoelectric. If pH under isoelectric condition,
then the charge will be positive charge. Meanwhile, if pH upper isoelectric condition,
then the charge will be negative charge or netral.
The reaction is :
COO- COOH
+
H
H3N + C H H3N + C H
R R
Base condition
First step is take 3 ml of NaOH 0,1M, then entered into test tube. After that
added several drops of PP indicatore. After added PP, the solution become pink
solution. PP indicatore have pH average about 8,3 – 10,0. If with acid, then not
changes and still colorless, but if with base, will be changes become pink solution. So,
NaOH is truthly base compund and this mixture used to as compared.
Protein 42
First take 2 ml of protein solution (egg) then entered into test tube. Next,
added drop by drops of NaOH 0,1 M colorless. After that observed, and resulting
strong pink solution with little flake on bottom. The function of NaOH is to give base
condition and to increas pH isoelectric which can make protein have base
characteristic. PP indicator which added will bonded by base group NHI, that is causes
the solution become pink solution.
Milk
First take 2 ml of protein solution (milk) then entered into test tube. Next,
added drop by drops of NaOH 0,1 M colorless. After that observed, and resulting soft
pink color. The function of NaOH is to give base condition and to increas pH
isoelectric which can make protein have base characteristic. PP indicator which added
will bonded by base group NHI, that is causes the solution become pink solution.
The reaction is :
COO- COOH
-
OH
H3N + C H H3N + C H
R R
For thirth experiment is about Precipitation of proteins. In this experiment
separated by three experiment, there are with ammonium sulphate, with mineral acid,
and with heavy metal.
With ammonium sulphate
Take 2 ml of egg solution then entered into test tube. Then, added 2 ml of
saturated ammonium sulphate. Shaken it with slowly until turbid and little white
precipitate. After that added 1 ml of aquadest until become colorless solution and little
flake. This experiment is reversible. The function of ammonium sulphate is to
precipitating proteins, but when it added by more, will dilute precipitate of proteins.
Added by ammonium sulphate causes hidraze protein, so litlle soluble protein will
precipitating. The proteins precipitated not occur chemical changes, so easy to
dissolve in water.
Protein 43
Milk
Take 2 ml of egg solution then entered into test tube. Then, added 2 ml of
saturated ammonium sulphate. Shaken it with slowly until white and little precipitate.
After that added 1 ml of aquadest until become colorless in bottom and white solution
on top. This experiment is reversible. The function of ammonium sulphate is to
precipitating proteins, but when it added by more, will dilute precipitate of proteins.
Added by ammonium sulphate causes hidraze protein, so litlle soluble protein will
precipitating. The proteins precipitated not occur chemical changes, so easy to
dissolve in water.
The reaction is :
With mineral acid

This experiment separated again become two kind of mineral acid, there are
HNO3 and HCl concentrate.
With HNO3
Take 1 ml of HNO3 then entered into test tube. Tilted the tube, after that
added 1 ml of egg solution drop by drops through tube wall. Let it stand until the
white ring formed as protein precipitate. But, if it cooled, the ring will changes
become yellow color. Then, shaken it. After that added HNO3 again and make the
precipitate more over than before. This experiment is irreverrsible because added by
HNO3. HNO3 concentrate causes of formed salt compound by acid reaction with
amino group of proteins.
Milk
Take 1 ml of HNO3 then entered into test tube. Tilted the tube, after that
added 1 ml of milk drop by drops through tube wall. Let it stand until the white ring
formed as protein precipitate. But, if it cooled, the ring will changes become little
yellow color under ring. Then, shaken it. After that added HNO3 again and make the
precipitate more over than before. This experiment is irreverrsible because added by
HNO3. HNO3 concentrate causes of formed salt compound by acid reaction with
amino group of proteins.
Protein 44
The reaction is :
With HCl
Take 1 ml of HCl concentrate then entered into test tube. Tilted the tube,
after that added 1 ml of egg solution drop by drops through tube wall. Let it stand
until the white ring formed as protein precipitate. Then, shaken it. After that added
HCl again and make the egg solution become colorless. Count of drops which
needed about 30 drops. This experiment is reverrsible because added by HCl. HCl
concentrate causes of proteins denaturazed by result precipitate that easy to dissolve
again.
Milk
Take 1 ml of HCl concentrate then entered into test tube. Tilted the tube, after
that added 1 ml of milk drop by drops through tube wall. Let it stand until the white
ring formed as protein precipitate. Then, shaken it. After that added HNCl again and
make the milk become colorless. Count of drops which needed about 200 drops. This
experiment is reverrsible because added by HCl. HCl concentrate causes of proteins
denaturazed by result precipitate that easy to dissolve again.
The reaction is :
With heavy metal

The principle of the reaction is netralizing charges. The precipitation can
occur if protein in isoelectric from that have negative charge. With positive charge of
heavy metal, will occur netralizing reaction by protein and resulted netral salt protein
which precipitating.. protein precipitate will back to dissolve when added by alkalyne
(as NH3 and NaOH). This characteristic of protein precipitations is reverrsible.
Protein 45
In this experiment, do in five kind of heavy metal. Consist of CuSO4, PbSO4,
ZnSO4, FeSO4 and HgSO4.
For CuSO4
Take 1 ml of egg solution, entered into test tube. Then added CuSO4 drop by
drops (need 3 drops when experiment which have do it). The color CuSO4 is light blue
solution. Shaken it until formed blue precipitate. The precipitate can formed caused by
protein power or amino acid to bonded by metal ions on the upper isoelectric point.
This power causes if pH on the upper of isoelectric point or amino acid will negative
charges. That is so can the proteins bonding metal ions which have positive ions.
Next step is the precipitate which formed, take the sample of it and move into
other test tube. After that, the sample added CuSO4 drop by drop (need 5 drops when
experiment which have do it) to dilute the precipitate (the sample) and result blue
solution.
Milk
Take 1 ml of milk, entered into test tube. Then added CuSO4 drop by drops
(need 3 drops when experiment which have do it). ). The color CuSO4 is light blue
solution. Shaken it until formed little blue precipitate, but the precipitate which
resulted just little. The precipitate can formed caused by protein power or amino acid
to bonded by metal ions on the upper isoelectric point. This power causes if pH on the
upper of isoelectric point or amino acid will negative charges. That is so can the
proteins bonding metal ions which have positive ions.
other test tube. After that, the sample added CuSO4 drop by drop (need 20 drops when
experiment which have do it) to dilute the precipitate (the sample) and result blue
solution.
The reaction is :
O O
R2 R2 + SO42- (aq)
H N CH C + CuSO4 (aq) H
Cu 2 N CH C
2 2 N C COO
N C COO- R H
R H (aq) H 2 (s)
H
For PbSO4
Take 1 ml of egg solution, entered into test tube. Then added PbSO4 drop by
drops (need 3 drops when experiment which have do it). The color of PbSO4 is
Protein 46
colorless. Shaken it until formed white precipitate but the precipitate very little. The
precipitate can formed caused by protein power or amino acid to bonded by metal
ions on the upper isoelectric point. This power causes if pH on the upper of
isoelectric point or amino acid will negative charges. That is so can the proteins
bonding metal ions which have positive ions.
Next step is the precipitate which formed, take the sample of it and move
into other test tube. After that, the sample added PbSO4 drop by drop (need 3 drops
when experiment which have do it) to dilute the precipitate (the sample) and result
colorless solution.
Milk
Take 1 ml of milk, entered into test tube. Then added PbSO4 drop by drops
(need 3 drops when experiment which have do it). The color of PbSO4 is colorless.
Shaken it until formed white precipitate, but the precipitate which resulted just little.
The precipitate can formed caused by protein power or amino acid to bonded by
metal ions on the upper isoelectric point. This power causes if pH on the upper of
isoelectric point or amino acid will negative charges. That is so can the proteins
bonding metal ions which have positive ions.
other test tube. After that, the sample added PbSO4 drop by drop (need 3 drops when
experiment which have do it) to dilute the precipitate (the sample) and the result is
white solution.
The reaction is :
O O
R2 R2 + 2 OAc- (aq)
H N CH C + Pb(OAc)2 (aq) Pb H2N CH C
2 2 N C COO
N C COO- R H
R H (aq) H 2 (s)
H
For ZnSO4
Take 1 ml of egg solution, entered into test tube. Then added ZnSO4 drop by
drops (need 3 drops when experiment which have do it). The color ZnSO4 is
colorless solution. Shaken it until formed white precipitate. The precipitate can
formed caused by protein power or amino acid to bonded by metal ions on the upper
isoelectric point. This power causes if pH on the upper of isoelectric point or amino
acid will negative charges. That is so can the proteins bonding metal ions which have
positive ions.
Protein 47
into other test tube. After that, the sample added ZnSO4 drop by drop (need 3-5 drops
turbid solution.
Milk
Take 1 ml of milk, entered into test tube. Then added ZnSO4 drop by drops
(need 3 drops when experiment which have do it). ). The color ZnSO4 is colorless
solution. Shaken it until formed white precipitate, but the precipitate which resulted
just little. The precipitate can formed caused by protein power or amino acid to
bonded by metal ions on the upper isoelectric point. This power causes if pH on the
into other test tube. After that, the sample added ZnSO4 drop by drop (need 3-5 drops
colorless solution.
The reaction is :
O O
R2 R2 + SO42- (aq)
+ ZnSO4 (aq) Zn H2N CH C
2 H2N CH C N C COO
N C COO- R H
R H (aq) H 2 (s)
H
For FeSO4
Take 1 ml of egg solution, entered into test tube. Then added FeSO4 drop by
drops (need 3 drops when experiment which have do it). The color FeSO4 is light
orange solution. Shaken it until formed orange precipitate. The precipitate can formed
caused by protein power or amino acid to bonded by metal ions on the upper
isoelectric point. This power causes if pH on the upper of isoelectric point or amino
acid will negative charges. That is so can the proteins bonding metal ions which have
positive ions.
other test tube. After that, the sample added FeSO4 drop by drop (need 3-5 drops when
experiment which have do it) to dilute the precipitate (the sample) and result orange
solution.
Protein 48
Milk
Take 1 ml of milk, entered into test tube. Then added FeSO4 drop by drops
(need 3 drops when experiment which have do it). ). The color FeSO4 is orange
other test tube. After that, the sample added FeSO4 drop by drop (need 3-5 drops when
experiment which have do it) to dilute the precipitate (the sample) and result light
white-yellow solution.
The reaction is :
O O
R2 R2 + SO42- (aq)
+ FeSO4 (aq) Fe H2N CH C
2 H2N CH C N C COO
N C COO- R H
R H (aq) H 2 (s)
H
For HgSO4
Take 1 ml of egg solution, entered into test tube. Then added HgSO4 drop by
drops (need 3 drops when experiment which have do it). The color HgSO4 is colorless
solution. Shaken it until formed white precipitate. The precipitate can formed caused
by protein power or amino acid to bonded by metal ions on the upper isoelectric point.
This power causes if pH on the upper of isoelectric point or amino acid will negative
charges. That is so can the proteins bonding metal ions which have positive ions.
other test tube. After that, the sample added HgSO4 drop by drop (need 3-5 drops
colorless with little flake.
Milk
Take 1 ml of milk, entered into test tube. Then added HgSO4 drop by drops
(need 3 drops when experiment which have do it). ). The color HgSO4 is colorless
Protein 49
other test tube. After that, the sample added HgSO4 drop by drop (need 3-5 drops
turbid solution.
The reaction is :
O O
R2 R2 + SO42- (aq)
+ HgSO4 (aq) Hg H2N CH C
2 H2N CH C N C COO
N C COO- R H
R H (aq) H 2 (s)
H
4. Color Reaction of Protein

a. Biuret Reaction
Egg
In this experiment the first time was to take 3 mL of egg white protein
solution that was slightly yellowish and put into a test tube. Then added 1 mL of
NaOH 40% colorless solution. Once added NaOH is produced a colorless solution
on the test tube. Thereafter, 0.5% CuSO4 solution was added with a solution of
blue color dropwise and required 4 drops of CuSO4 to produce a purple solution
which showed a positive reaction to the biuret test. The function of the addition of
NaOH is to provide an alkaline atmosphere to the protein. While the addition of
CuSO4 serves to determine the existence of peptide bonds or not in protein
samples. With the formation of purple color in this protein is due to the formation
of complex compounds between Cu2 + and N of peptide bond molecules. Where
more and more amino acids on the peptide bonds the purple color gets thicker.
Milk
In this experiment the first time was to take 3 ml of white milk protein
solution and put into a test tube. Then added 1 mL of NaOH 40% colorless
solution. After added NaOH the resulting solution remains white in the test tube.
Thereafter, 0.5% CuSO4 solution was added in the form of blue solution dropwise
and required 3 drops of CuSO4 to produce a purple solution which showed a
positive reaction to the biuret test. The function of the addition of NaOH is to
provide an alkaline atmosphere to the protein. While the addition of CuSO4 serves
to determine the existence of peptide bonds or not in protein samples. With the
formation of purple color in this protein is due to the formation of complex
Protein 50
compounds between Cu2+ and N of peptide bond molecules. Where more and more
amino acids on the peptide bonds the purple color gets thicker.
Biuretic reactions are a common color reaction to test for the presence of
peptide groups in proteins. The positive reaction is characterized by the formation
of purple color due to the formation of complex compounds between Cu2+ and N
from peptide bond molecules. In our experiment, the egg white protein solution
yielded a deeper purple color than milk protein, so the egg protein contains more
peptides than milk proteins. The equation of the reaction is
CuSO4 (aq) + 2 NaOH (aq) → Cu(OH)2 (aq) + Na2SO4 (aq)
Cu(OH)2 (aq) → Cu2+ +2OH-
Cu2+
b. Xantoprotein reaction
Egg
In this experiment the first time was to take 3 ml of egg white protein
solution and put into the test tube. Then 1 mL concentrated HNO3 concentrated
solution was added. After the added concentrated HNO3 produced a white
sediment and a little yellow colored solution. Then the test tube is heated to a
water bath and produces a yellow solution and there is a spreading white sediment.
In this experiment, nitration reacts to the benzene nucleus of amino acids found in
protein molecules by nitro groups. The nucleus of benzene can be nitrated by
concentrated HNO3 and produce nitrobenzene derivatives. So the addition of
concentrated HNO3 was functioned to minify the benzene core of amino acids. The
next step is the reaction tube allowed to cool. Then after cool, NH3 added dropwise
and produced an orange solution. To produce orange color is required as much as 2
drops of NH3. On the addition of NH3 the yellow color turns to orange. This is due
to the acidity of phenol reacting with ammonia.
Milk
In this experiment the first time was to take 3 ml of white milk protein
solution and put into a test tube. Then 1 mL concentrated HNO3 concentrated
Protein 51
solution was added. After the added concentrated HNO3 produced a white
sediment and a little yellow colored solution. Then the test tube is heated to a
water bath and produces a yellow solution and there is a spreading white sediment.
In this experiment a nitration reaction occurs at the benzene nucleus of the amino
acid present in the protein molecule by the nitro group. The nucleus of benzene
can be nitrated by concentrated HNO3 and produce nitrobenzene derivatives. So
the addition of concentrated HNO3 was functioned to minify the benzene core of
amino acids. The next step is the reaction tube allowed to cool. Then after cool,
NH3 added dropwise and produced an orange solution. To produce the color
orange NH3 required as much as 5 drops. On the addition of NH3 the yellow color
turns to orange. This is due to the acidity of phenol reacting with ammonia.
From this experimental test of xanthoprotein, the positive result is
indicated by the yellow color caused by the nitration reaction at the benzene
nucleus of the amino acid so that the formation of the polynitrobenzene compound
and after the ammonia is added the color becomes orange because the acidity of
the phenol reacts with ammonia. On the addition of the yellow alkali compound
will disappear and turn into orange due to the acidity of the phenol reacts with the
alkali. The equation of the reaction is
H2 H H2 H
C C COOH + HNO3 C C COOH
NH2 NH2
NO2
fenilalanin fenilalanin ternitrasi (kuning)
c. Nynhidrin Reaction
Egg
solution that was slightly yellowish and put into a test tube. The pH is then
adjusted to a precise pH of 7 which is neutral in a way tested with a universal
indicator. Add a solution of NaOH if the protein solution is too acidic and add
acetic acid solution if it is too alkaline. Thereafter, 10 drops of 0.2% ninhydrin
solution were added. After the addition of ninhydrin solution produced a solution
of colorless. Then the test tube is heated over a water bath at 100° C for 10
minutes or until a reaction occurs. By heating the resulting purple solution to form
a purple ruheman complex. So the protein in the egg contains α-amino acids.
Milk
Protein 52
In this experiment first performed is to take 1 mL of milk protein solution
that is white and put into a test tube. The pH is then adjusted to a precise pH of 7
which is neutral in a way tested with a universal indicator. Add a solution of
NaOH if the protein solution is too acidic and add acetic acid solution if it is too
alkaline. Thereafter, 10 drops of 0.2% ninhydrin solution were added. After the
addition of ninhydrin solution produced a white solution faded. Then the test tube
is heated over a water bath at 100 ° C for 10 minutes or until a reaction occurs. By
heating the resulting purple-colored solution forming a purple ruheman complex.
So milk protein contains α-amino acid. In the ninhydrin reaction, a positive result
is characterized by the formation of a purple or blue solution after heating,
indicating that there is an α-amino acid content in the protein. Amino acids react
with ninhydrin to form aldehydes with one lower C atom and release NH3 and CO2
molecules Ninhydrin will be reduced and react with NH3. The equation of the
reaction is
O
OH
pH = 7
H CH NH2 +
OH 100oC
COOH O
Ninhidrin
O O
N +R COH + CO2 + H2O
O O
kompleks ungu Ruheman (biru-ungu)
d. Millon Reaction
Egg
In this experiment, the first attempt was to take 2 mL of egg white protein
solution which was slightly yellowish and put into a test tube. Then 1 mL of
colorless millon reagent was added. After added reagents are produced a solution
of colorless. Then heated the reaski tube in a water bath and formed a red
sediment. then added 1% NaNO2 and reheated. obtained red color with red
sediment. This suggests that the proteins in eggs contain tyrosine / tryptophan and
also phenol group
Milk
Protein 53
In this experiment the first attempt was to take 2 ml of white egg white
protein solution and put into a test tube. Then 1 mL of colorless millon reagent
was added. After the reagents are added the resulting solution remains white. Then
heated the reaski tube in a water bath and formed a red sediment. Then added 1%
NaNO2 and reheated. obtained red color with red sediment. This suggests that the
proteins in eggs contain tyrosine / tryptophan and also phenol groups.
If the protein is reacted with millon will be a positive value marked by the
formation of a red complex. At first Hg dissolved in HNO3 oxidized to Hg+. This
Hg+ ion further forms a salt with a carboxyl group of tyrosine. When heated the
red sediment formed. This happens because the nitric acid that originally serves as
a solvent oxidizes Hg+ to Hg2+. At the same time, the amino acid tyrosine
ternitrasi. Then the reaction of the formation of HgO red. The formation of the red
complex shows the presence of tyrosine / tryptophan and also phenol compounds
in milk and egg whites proteins. The equation of the reaction is
H HNO3 H Hg2+
HO C COOH HO C COOH
NH2 O N NH2
H NH2
N O
O O
H2 C CH C OH
H
HO C C C Hg2+ H2
O O N O
H O
NH2
kompleks merah
e. Hopkine-Cole Reaction
Egg
solution that was slightly yellowish and put into a test tube. Then added 1 drops of
dilute formaldehyde that is not colored. And a colorless solution was produced on
the test tube. Thereafter, 1 drop of mercury sulphate reagent was added to colorless
And the resulting solution remains colorless. Then 1 mL of concentrated H2SO4 is
added through the tilt reaction tube wall. After the addition of concentrated H2SO4
produced a du layer of white sediment and brown solution where the white
deposits that are above the grey solution. This is not in accordance with the theory
Protein 54
that will form two layers, which is visible purple ring and brownish purple
solution.
Milk
In this experiment first performed is to take 1 mL of milk protein solution that is
white and put into a test tube. Then added 1 drops of dilute formaldehyde that is
not colored. And the resulting solution remains white in the test tube. Thereafter, 1
drop of mercury sulphate reagent was added to yellow. And the resulting solution
remains white. Then 1 mL of concentrated H2SO4 is added through the tilt reaction
tube wall. After the addition of concentrated H2SO4 produced a du layer of white
sediment and a solution of gray. This is not in accordance with the theory that will
form two layers, which is visible purple ring and brownish purple solution.
The positive result of the reaction in this experiment was the formation of a
purple ring on the boundary plane between the protein solution and the reagent.
The formation of this ring is due to the formation of condensation of 2 indol cores
from tryptophan with aldehyde groups of glyoxylic acid in the atmosphere of
sulfuric acid. Tryptophan is the only amino acid that contains the indole group. If
the reaction results indicate a purple ring, the protein contains the indole nucleus
of tryptophan with aldehyde. The equation of the reaction is
O
H H
CH2 CHCOOH
C
H COOH
+
NH 2 O NH2
N
C N
H H
asam amino triptofan H
asam glioksilat .
5. Hydrolisis of Protein
Egg solution
In this experiment the first step is to insert 1 mL of a slightly yellowish colored
egg protein into the test tube. Then added 1 mL of NaOH 40%. In the test tube
produced a turbid solution. The next step is the reaction tube heated above the water
bath for 1 minute. Produced an increasingly murky solution. Then added 1 drops of
colorless PbSO4. The reaction tube produced a blackish solution (turbid). The function
of adding NaOH is to hydrolyze the peptide bonds of the protein polymer and to the
hydrolysis to produce the amino acid monomer. In a protein solution it produces a
Protein 55
browm blackish color this is because the S atom reacts acetic acid and forms a PbS
precipitate.
Milk
In this experiment the first step is to insert 1 mL of white milk protein solution
into the test tube. Then added 1 mL of NaOH 40%. In the test tube produced a white
solution. The next step is the reaction tube heated above the water bath for 1 minute.
And the resulting solution is yellow (white top layer). Then, the test tube was added 1
drop of colorless PbSO4. Produce a green brownish. The function of adding NaOH is
to hydrolyze the peptide bonds of the protein polymer and to the hydrolysis to produce
the amino acid monomer. In a protein solution it produces a blackish color this is
because the S atom reacts acetic acid and forms a PbS precipitate.
Pb2+(aq) + 4OH-(aq) → PbO22-(aq) + H2O(l)
S2-(aq) + 2H2O(aq) + PbO22-(aq) → PbS↓(s) + 4OH-(aq)

IX. Discussion
In Hopkin-cole reaction experiments there is a mismatch between the results and
the theory. Based on the theory, proteins in egg white and milk are formed 2 layers
(purple ring). However in our experiment we did not form a purple ring. This is because
the reagent H2SO4 used is not in good condition or has been contaminated so as not to be
able to get the results that should be.
X. Conclusion
Based on the experiments we have done, it can be concluded that:
1. Protein in milk and eggs can be denatured by acid addition, this is indicated by the
presence of white precipitate in both protein solutions.
2. Proteins may be denatured by heating. Denaturation of milk and egg proteins is
indicated by the presence of white precipitate.
3. Protein in milk and eggs denatured due to the addition of formadehid compounds.
Denaturation is irreversible characterized by the presence of white precipitate.
4. Proteins can react with acids and can also react with bases. Therefore it can be
concluded that the protein is amphoteric.
5. Protein will precipitate by adding solution (NH4)2SO4 and soluble precipitate with
addition of reversible aquadest.
6. Precipitation with HNO3 is irreversible while precipitation with HCl is reversible.
7. Precipitation with heavy metals is reversible. Precipitation occurs when the protein is
in a negatively charged isoelectric form.
Protein 56
8. In the color test of proteins with biuret reactions, the results show that egg protein and
milk contain peptides. As indicated by the change of color to purple (complex)
9. The addition of NaOH will hydrolyze the peptide bonds of the protein polymer and
produce amino acid monomers. In a protein sample containing sulfur it is
characterized by a blackish-colored solution indicating the presence of PbS precipitate.
10. In the color test of proteins by reaction ksanthoprotein, showed the results that the
protein of eggs and milk formed of polynitro benzene compounds of amino acids
characterized by the presence of yellow.
11. In the color test of proteins with the reaction of ninhydrin, showed that in egg protein
and milk containing amino acids characterized by the formation of blue-violet color
12. In the color test of proteins by millon reaction, showed that in egg protein and milk
containing phenol group characterized by red sediment
13. In the color test of proteins with hopkin-cole reactions, it shows that egg protein and
milk proteins not contain the indole nuclei of tryptophan with aldehydes characterized
by the not formation of purple rings and brownish-purple solutions.
XI. Answer Question

1. Explain what is the function of testing the protein with each test reagent (CuSO4,
HgCl2, HNO3, Pb-acetate)!
Answer:
The function of protein test with each reagent as follows:
 CuSO4 is used to test for the presence of heavy metals in proteins characterized by
presence of precipitation when positive proteins contain heavy metals.
 HgCl2 is used for the test of a protein containing a phenyl hydroxyl group (-OH).
 HNO3 is used to test for the presence of benzene rings from the amino acid salt of
the proteins, ie in this experiment when concentrated nitric acid is added and
produces nitrobenzene derivatives.
 Acetate pb is used to test for the presence of cysteine and methionine amino acids,
which in this experiment will produce a black solution because the S atom reacts
with acetic acid to form PbS precipitate.
2. How does the effect of organic solvents (acetone and ethanol) on the nature of protein
denaturation?
Answer:
Protein 57
The effect of organic solvents (acetone, ethanol) on the nature of protein
denaturation is that proteins or nucleic acids will lose their secondary and tertiary
structures because organic solvents result in denatured proteins.
3. List the various bonds that cause the polypeptide to be stable in alpha-helical form!
Answer:
 Disulfide bond
Formed between 2 cysteine residues interconnected 2 parts of the
polypeptide chain through cysteine residues.
 . Hydrogen bond
Formed between the NH- or -OH groups and the C = O groups in the
peptide or -COO-bond in the R group.
XII. References
Anna, P. 1994. Dasar-Dasar Biokimia. Jakarta : Universitas Indonesia.
Anwar, Chairil. 1996. Pengantar Praktikum Kimia Organik. Jakarta : DIKTI.
Carey, Francis. 2000. Organic Chemistry. United State:McGrawHill.
Fessenden, Ralp J dan Joan S Fessenden. 1986. Kimia Organik jilid 2. Jakarta : Erlangga.
Hidajati, Nurul, dkk. 2017. Penuntun Praktikum Kimia Organik II. Surabaya:
Jurusan Kimia FMIPA Unesa.
McMurry, John. 2008. Organic Chemstry. United State of America : Thomson Learning
Inc.
Ophart, C.E., 2003. Virtual Chembook. Elmhurst College.
Poedjiaji A and Supriyanti. 2007. Dasar-Dasar Biokimia. Jakarta: UI Press
Protein 58
Wade, L G. 2013. Organic Chemistry. United State of America: Pearson Education.
XIII. ATTACHMENT
1. Denaturation of Proteins
Protein solution that used is egg

Prepare the tools for all Prepare the materials for all
and milk, each solution which
experiment experiment
needed is 5 ml
Formed white precipitate after After heated, egg solution become Prepare 2 -3 ml proteins solution
added 2 drops of CH3COOH 1N turbid and little flake, milk solution (egg and milk)
is white
Protein 59
After heated 1 minutes, egg Devided into 2 test tubes, and each Formaldehyde + aquadest become
become turbid and little white one of them heated + (NH4)2SO4, colorless solution
flake, while milk white solution but each other just heated
Formaldehyde + aquadest + Egg

resulting turbid solution and little
flake, while and milk resulting
white solution and little white
precipitate
2. Amphoter characteristic of Protein
HCl + aquadest resulting Prepare the Congo indicatore Added Congo indocatore resulting
colorless solution soft blue solution
Protein 60
If added by egg result soft pink NaOH + PP indicatore result pink When egg added by NaOH result
solution with white precipitate, by solution colorless solution, while milk and
milk result pink solution with NaOH result white solution
white precipitate
When added by PP indicatore, in

egg tube become strong pink
solution with little flake, in milk
tube become soft pink color
Prepare the protein solution, use 2 When added by (NH4)2SO4, for After added aquadest and shaken,
Protein 61
ml of egg and milk egg result turbid solution and little for egg result colorless solution
white precipitate. Foor milk result and little flake. For milk result
white solution and little precipitate colorless in bottom and white
solution on top
HNO3 + egg formed white ring as When added HNO3 again into each HCl + egg formed white ring, HCl
precipitate, but for a few second tube, egg or milk still have + milk formed white ring too. The
become yellow ring. HNO3 + precipitate while little dissolve ring showing as precipitate
milk formed white ring and little
yellow color
When added HCl again into each Prepare protein (egg) solution 1 ml Also prepare the milk entered into
tube, egg will be colorless into 5 test tube 5 test tube each 1 ml
solution (30 drops) and milk will
be colorless solution too (200
drops)
Protein 62
Egg added by some heavy metal Milk added by some heavy metal Take sample from each test tube of
(CuSO4, PbSO4, ZnSO4, FeSO4 (CuSO4, PbSO4, ZnSO4, FeSO4 egg, then diluted with similar
and HgSO4) and HgSO4) heavy metal which added before
After add, all result precipitate, After add, all result precipitate, but CuSO4 (5 drops), PbSO4 (3 drops),
but for PbSO4 just little formed for PbSO4 just little formed ZnSO4 (3-5 drops), FeSO4 (3-5
precipitate precipitate drops), HgSO4 (3-5 drops)
Take sample from each test tube

of milk, then diluted with similar
heavy metal which added before
CuSO4 (20 drops), PbSO4 (3
drops), ZnSO4 (3-5 drops), FeSO4
(3-5 drops), HgSO4 (3-5 drops)
4. Reaction the Color of Protein
Protein 63
Prepare protein solution, egg and After added NaOH + CuSO4, for Prepare protein solution, egg and
milk egg solution result purple color milk
(++), for milk result purple, turbid
Egg solution + HNO3 result light After heated, egg result yellow and When added by ammonia
yellowish solution, milk + HNO3 white solution, for milk result concentrate, for egg result orange
result light yellowish solution yellow and white solution, turbid solution, for milk result orang
solution, turbid
Egg + Nynhidrin + heated result Prepare protein solution, egg and When added millon reagent, for
blue purplekish (++), while milk milk egg result turbid and colorless
+ nynhidrin + heated result blu solution, for milk result white
purplekish turbid solution
Protein 64
After heated, both of them result When added by NaNO2, for egg Prepare protein solution, egg and
red precipitate result red solution and red milk
precipitate, for milk result red
solution and little red precipitate
which float up
Egg + formaldehyde + Millon When egg added by H2SO4

reagent result white solution, concentrate result grey solution.
turbid. Wile milk + formaldehyde Milk added by H2SO4 concentrate
+ Millon reagent result white result grey solution in the bottom
solution turbid and white precipitate on the top
5. Protein Hydrolisis and Sulphur Identification
Egg + NaOH result colorles, milk After heated, egg become Egg + Pb acetic result brown
+ NaOH result white solution yellowish solution, while milk blackish solution. Milk + Pb acetic
turbid become yellow, turbid solution result reen brownish
Protein 65

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I.

Title of Experiment : Understanding Characteristic and Color Reaction of

Picture 1. Amino Acid

Picture 2. The example from Classification of amino acid

Picture 3. Structure in acid and base solution

Picture 5. Isoelectric Point (McMurry, 2008).

Picture 6. Esterification of an amino acid.

2. Reaction with Ninhydrin

Picture 8. Peptide Bond

Table 1. Classes of Conjugated Proteins.

 Test tube 20 pieces

- Entered into test tube

b. Denaturation because of heating

2-3 mL of protein solution

- Entered into test tube

Test tube 1 Test tube 2

c. Denaturation because of Formaldehyde

- Entered into test tube

b. Test in base condition

3 mL of NaOH 0,1 M 2-3 ml protein solution

- Entered into test tube. - Entered into test tube

b. Protein precipitation with mineral acid

- Poured into test tube

- Poured into test tube

c. Protein precipitation with heavy metal

1-1,5 mL Protein solution 1-1,5 mL Protein solution

it until precipitate dilute shaken it until precipitate dilute

4. Reaction the Color of Protein

- Entered into test tube

- Entered into test tube

5 mL protein turbid + white denaturation and formed adding acetic acid, it is

- Entered into test tube Aquadest : protein precipitate) and precipitate.

2. Amfoter Characteristic of Protein Aquadest : Aquadest + Protein in egg and milk is

b. Xanthoprotein reaction HNO3 Egg + HNO3 : Protein in egg and milk

+ heated : blue + + CO2 + H2O

1 ml protein little turbid solution S, because react with Pb-

With mineral acid

With heavy metal

4. Color Reaction of Protein

Cu(OH)2 (aq) → Cu2+ +2OH-

N +R COH + CO2 + H2O

S2-(aq) + 2H2O(aq) + PbO22-(aq) → PbS↓(s) + 4OH-(aq)

XI. Answer Question

Protein solution that used is egg

Formaldehyde + aquadest + Egg

When added by PP indicatore, in

Take sample from each test tube

Egg + formaldehyde + Millon When egg added by H2SO4

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