I

II
III

IV

Title
: Learning Characteristic and Reaction of Protein
Date of Experiment
: Thursday, September 15th 2016
Objective
:
a Differentiate the solubility properties of protein as reversible and irreversible.
b Differentiate the denaturation reaction of protein that caused by acid, salt, and
salt from hard metal and also the heating on observation.
c Understand the cause of precipitation in protein
d Identify the protein accurence based on the color reaction.
Basic Theory:
Protein is one of the giant biomolecules, besides polysaccharides, lipids, and
polynucleotides, which is the main constituent of living beings. In addition, the
protein is one of the most studied molecules in biochemistry. Protein discovered by
Jns Jakob Berzelius in 1838. The chemical can be distinguished between simple
protein consisting of the polypeptide and protein complexes containing substances
such as food additives Hern, carbohydrates, lipids or nucleic acids. For complex
proteins, polypeptides section called aproprotein and called haloprotein entirety.
Functionally protein also showed a lot of difference. In their cells function as
enzymes, building materials, lubricants and carrier molecules. But actually the
protein is a natural polymer composed of various amino acids by peptide bonds.
This is the structure of protein.

(Hart, 1987).
Most protein molecules retain their biological activity only within a very
limited range of temperature and pH. Exposing soluble or globular proteins to
extremes of pH or to high temperatures for only short periods causes most of them to
undergo a physical change known as denaturation, in which the most visible effect is
a decrease in solubility. Since no covalent bonds in the backbone of the polypeptide
chain are broken during this relatively mild treatment, the primary structure remains
intact.
Denaturation is the process of altering the shape of a protein without
breaking the amide bonds that form the primary structure. High temperature, acid,
base, and even agitation can disrupt the noncovalent interactions that hold a protein
in specific shape. Heat breaks up week London forces between nonpolar amino
acids. Heat, acid, and base disrupt hydrogen bonding interactions between polar
amino acids, which account for much of the secondary and tertiary structure. As a
result, denaturation causes a globular protein to uncoil into an undefined randomly
looped structure. Denaturation is caused by exposure to excessive heat or extreme
pH values (Vollhardt and Schore, 2011).

Most globular proteins undergo denaturation when heated above 60 to 70C.

Denaturation often makes globular protein less water soluble. Globular proteins are
typically folded with hydrophobic regions in the interior to maximize the interaction
of polar residues on the outside surface with water. This make them water soluble.
When the protein is denaturated, more hydrophobic regions are exposed and the
protein often loses water solubility (Smith, 2010).
Formation of an insoluble white coagulum when egg white is boiled is a
common example of protein denaturation. But the most significant consequence of
denaturation is that the protein usually loses its characteristic biological
activity; e.g., heating usually destroys the catalytic ability of enzymes.
Denaturation causes the shape changing of proteins. Changing of protein
configuration from -helix shape become longer (Tim Dosen Kimia Organik, 2016).
Denaturation is the unfolding of the characteristic native folded structure of the
polypeptide chain of globular protein molecules. When thermal agitation causes the
native folded structure to uncoil or unwind into a randomly looped chain, the protein
loses its biological activity. In certain cases denaturation of native proteins into
biologically inactive forms is not irreversible. An unfolded protein molecule
simetime may spontaneously returns to its native biologically active form in the test
tube, a process called renaturation. If the denatured protein was an enzyme, its
catalytic activity returns on renaturation, without change in the specificity of the
reaction catalyzed.
Protein denaturation is the partial or complete disorganization of protein's
characteristic three-dimensional shape as a result of disruption of its secondary,
ternary, and quaternary structural interactions. Because the biological function of
protein depends on its three-dimensional shape, the result of denaturation is loss of
biological activity. Protein denaturation does not affect the primary structure of
protein.
Although some proteins lose all of their three-dimensional structural
characteristics upon denaturation, most proteins maintain some three-dimensional
structure. For few small proteins, it is possible to find conditions under which the
effects of denaturation can be reversed; this restoration process in which the protein
is "refolded" is calledrenaturation. Denaturation is irreversible, however, for most
proteins.
most dramatic example of protein denaturation occurs when egg white (
concentrated solution of the protein albumin) is poured into hot surface. The clear
albumin solution immediately changes into white solid with jelly-like
consistency. similar process occurs when hamburger juices encounter hot
surface. brown, jelly-like solid forms.

Precipitation
Precipitation is widely used for product recovery of biomolecules especially
proteins. Precipitation is usually induced by addition of a salt or an organic solvent or
by changing the ph to alter the nature of the solution. Functional group (NH2, NH, OH,
CO) and zwitter ion in the structure of proteins can cause precipitation reaction of
protein (Tim Dosen Kimia Organik. 2016).
The most common type of precipitation for protein is salt induced
precipitation. Protein solubility depends on several factors. Proteins in solution show
profound changes in solubility as a function of (1) pH, (2) ionic strength, (3) the
dielectric properties of the solvent (hydrated shell), and (4) temperature.
The solubility of most globular proteins is profoundly influenced by the pH of
the system because the electric charge of protein molecule results from pH. When the
protein molecule has no net electric charge there is no electrostatic repulsion between
neighboring protein molecules and they tend to coalesce and precipitate. When all the
protein molecules have a net charge of the same sign they repel each other, preventing
coalescence of single molecules into insoluble aggregates.
Electric charge of proteins and hence the availability of hydrated shell and
solubility of proteins depend also on the ionic composition of the medium, since
proteins can bind certain anions and/or cations (Girinda, 1990).
Methods of protein precipitation.
There are two methods of protein precipitation: reversible (salting-out) and
inreversible (denaturation).
Reversible coagulation of proteins. Salting-in and Salting-out of Proteins.
Reversible coagulation of proteins - precipitation without the loss of native
structure. If optimal conditions will be created for proteins (for example, the adding of
solvent) they can be dissolved again.
Neutral salts have pronounced effects on the solubility of globular proteins. In
low concentration, salts increase the solubility of many proteins, a phenomenon called
salting-in. Salts of divalent ions, such as MgCl2 are far more effective at salting-in
than salts of monovalent ions, such as NaCl and KCl. The ability of neutral salts to
influence the solubility of proteins is a function of their ionic strength, a measure of
both the concentration and the number of electric charges on the cations and anions
contributed by the salt. Salting-in effects are caused by changes in the tendency of
dissociable R groups on the protein to ionize.
On the other hand, as the ionic strength is increased further, the solubility of a
protein begins to decrease. At sufficiently high ionic strength a protein may be almost
completely precipitated from solution, an effect called salting-out. The
physicochemical basis of salting-out is rather complex; one factor is that the high
concentration of salt may remove water of hydration from the protein molecules, thus
reducing their solubility, but other factors are also involved. Proteins precipitated by

salting-out retain their native conformation and can be dissolved again, usually
without denaturation. Ammonium sulfate is preferred for salting out proteins because
it is so soluble in water that very high ionic strengths can be attained (Young, 1994).
Qualitative Reactions on The Proteins and Amino Acids
1

Biuret test
The protein is warmed gently with 10 % solution of sodium hydroxide and
then drop of very dilute copper sulphate solution is added, the formation of
reddish- violet colour indicates the presence of peptide link, NH . The
test is given by all proteins, peptones and peptides. Its name is derived from the
fact that the test is also positive for the compound biuret, 2N CONH
CONH2 obtained from urea by heating.
It should be noted that dipeptides do not give the biuret test, while all other
polypeptides do so. Hence biuret test is important to know whether hydrolysis of
proteins is complete or not. If the biuret test is negative, hydrolysis is complete, at
least to the dipeptide stage.

Xanthoproteic test
On treatment with concentrated nitric acid, certain proteins give yellow
colour. This yellow colour is the same that is formed on the skin when the latter
comes in contact with the concentrated nitric acid. The test is given only by the
proteins having at least one mole of aromatic amino acid, such as tryptophan,
phenylalanine, and tyrosine which are actually nitrated during treatment with
concentrated nitric acid.
Millon's test
Protein on adding Millon's reagent ( solution of mercuric and mercurous
nitrates in nitric acid containing little nitrous acid) followed by heating the
solution give red precipitate or colour. The test is responded by the proteins
having tyrosine. The hydroxyphenyl group of tyrosine is the structure responsible
for this test. Moreover, the non-proteinous material having phenolic group also
responds the test.
Foll reaction. This reaction reveals the sulfur containing amino acids
(cysteine, cystine). Treatment of the sulfur containing amino acids with salt of lead
and alkali yields a black sediment.

Adamkevich reaction
This reaction detects the amino acid tryptophan containing indol ring. The
addition of the concentrated acetic and sulfuric acids to the solution of tryptophan
results in the formation of red-violet ring appearing on the boundary of different
liquids.
Ninhydrin test
The ninhydrin colour reaction is the most commonly test used for the
detection of amino acids. This is an extremely delicate test, to which proteins, their
hydrolytic products, and -amino acids react. Although the test is positive for all
free amino groups in amino acids, peptides, or proteins, the test is much weaker for
peptides or proteins because not as many free groups are available as in amino

acids. For certain amino acids the test is positive in dilutions as high as 1 part in
100,000 parts of water.
When ninhydrin is added to protein solution and the mixture is heated to
boil, blue to violet colour appears on cooling. The colour is due to the formation of
complex compound.

The test is also given by ammonia, ammonium salts, and certain amines.
Ninhydrin is also used as reagent for the quantitative determination of free
carboxyl groups in solutions of amino acids.
6

Nitroprusside test
Proteins containing free -SH groups (of cysteine) give reddish colour with
sodium nitroprusside in ammonical solution.

Proteins are polypeptides that contain more than 50 amino acid units. The
dividing line between polypeptide and protein is arbitrary. The important point is
that proteins are polymers containing large number of amino acid units linked by
peptide bonds. Polypeptides are shorter chains of amino acids. Some proteins have
molecular masses in the millions. Some proteins also contain more than one
polypeptide chain (Young, 1994).
V

Tools And Materials

a. Tools
Beaker glass
100 mL and 50 mL
Graduated cylinder
10 mL and 25 mL
Test tube
Spatula
Bunsen bunner
Screen
Tripod
Pipete
Test tube rack
Electrical stove
b. Materials
Pure cows milk
Duck egg
CH3COOH 1 N solution
(NH4)2SO4 solution
Formaldehyde solution
HNO3 concentrated solution
HCl concentrated solution

2 pieces
2 pieces
20 pieces
1 piece
1 piece
1 piece
1 piece
15 pieces
1 piece
1 piece

 CuSO4 solution
PbSO4 solution
NaOH 40 % solution
CuSO4 0,5 % solution
Ninhydrin solution
Millon reagent
NaNO2 1% solution
PbSO4 solution
Aquades
PP indicator
Kongo indicator
FeSO4 solution
VI
Procedures
VII
Observation result
VIII Analysis and Explaning
IX
Conclusion
1 Denaturation of protein can be caused by react with acid, heating in high
temperature, and chemical compound addition.
2 Protein can react with acid and base (ampotheric characteristic).
X
References
Hart, H. 1987. Kimia Organik. AlihbBahasa : Sumanir Ahmadi. Jakarta: Erlangga.
Girinda, A. 1990. Biomchemistry. New York : Printia Hall.
Smith, Janice G. 2010. General, Organic, & Biological Chemistry. New York:
McGraw-Hill.
Tim Dosen Kimia Organik. 2016. Penuntun Praktikum Kimia Organik II.
Surabaya: Jurusan Kimia FMIPA Unesa.
Vollhardt, Peter and Neil Schore. 2011. Organic ChemistryStructure and function
sixth edition. New York: W.H. Freeman and Company.
Young, Derek R. 1994. Protein. (Online), (http://www.rpi.edu/dept/chemeng/biotech-environ/precip/preceintro.html , accessed on 11st september
2016 at 21.05 WIB).
XI
XII

Answering question
Attachment