Practicum Report

Biokimia Umum

Day/Date : Thursday /17th March 2016

Time
: 1500-1730
PJP
: Syaefudin, SSi, MSi
Asisten : 1. Annisa Dhiya Ak
2. M.Maftuchin S
3. Bayu Cakra

PROTEIN II
Nishalini A/P Magis Paran

B04158008

DEPARTEMEN BIOKIMIA
FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM
INSTITUT PERTANIAN BOGOR
BOGOR
2016

INTRODUCTION
Proteins are long chains of molecules composed of amino acids joined by peptide
bonds. Protein is needed by the body in a large amount and derived from foods
that are being consumed. Protein that originated from animals, such as fish, meat,
eggs, and milk are called animal proteins, whereas proteins that derived from
plants, such as beans called vegetable protein (Ariningsih & Rachman, 2008).
Protein is a nutrient that is essential for the body because it serves as protein
regulators, as a source of energy, and building blocks for body.
Based on the structure of the protein, it can be divided into four types, namely
primary, secondary, tertiary, and quaternary. The primary structure is twodimensional structure that describes the sequence of amino acids making up the
protein residues and backbone of peptide bond. Secondary structure is the result
of the folding of polypeptides resulted by hydrogen bonds with the carboxyl O-Namino group of a peptide bond. In this secondary structure, there are two types of
proteins, namely helix and folded sheets. Tertiary structure that is threedimensional structure resulted from the interaction of the polypeptide folding
between R groups. Tertiary structure can occur in the presence of hydrogen bonds,
disulphide bonds, salt bridges, and hydrophobic interactions. Quaternary structure
is the structure resulting from the interaction of tertiary structure with other
compounds, with protein or non-protein (McMurry, 2008).
The sample of protein that was used in the experiments is albumin. Albumin is a
serum or protein found abundantly in the body, reaching 2/3 of the total protein in
the body. The function of albumin is for formation of new cells and tissues as well
as to recover body tissues that has been damaged. Moreover, albumin also
transports materials that are less soluble in water passing through the blood
plasma and cell liquid (Indradjaja et al, 2014).
The characteristics of the protein to be observed in this experiment are the ability
of the protein to precipitate, coagulate and denature. Proteins may precipitate if
certain substances added, such as heavy metals and anorganic salts. The aim of
this experiment was to analyse and observe characteristic of proteins such as
precipitation of proteins by metals and salts, protein coagulation (thermal effect),
and protein denaturation.
Method
Time and Place of Practicum
Protein II practicum was held in Laboratorium B Biokimia, Departemen
Biokimia, Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Pertanian
Bogor. The time of practicum was at Thursday, 17th March from 1500 to 1730.
Aim of the Practicum
The objective of this experiment is to observe the effects of heavy metals, salt,
high temperature, alcohol, and pH towards protein solution.

Materials and Apparatus

Materials used in this experiment was 2 % HgCl2 solution, 5% Pb-acetate
solution, 5 % AgNo3 solution, albumin solution, (NH4)2SO4 salt, Biuret Reagent,
distilled water, 95% ethanol solution, 4.7 pH acetate buffer, 0.1M HCL, and 0.1 M
NAOH. The apparatus used were test tube, black bulb, pipette, filter paper, filter
funnel, water bath, test tube clamp, beaker, glass rod, and dropper.
Procedure
Protein precipitation by metal. 1.5 ml albumin solution is put into a test tube
and then 3 drops of 2% HgCl2 was added. Reactions that occur was observed. The
experiment was repeated using 5% Pb-acetate and 2% AgNO3 solution.
Protein precipitation by salt. 5 ml albumin solution was put into a test tube and
(NH4)2SO4 added to the solution little by little, it was stirred until it became
homogenous and then it was filtered, producing filtrate and precipitate. The
precipitate was dissolved in water. They both were tested with Biuret reagent.
Coagulation test. 5 ml of albumin solution was put into a test tube then boiled for
5 minutes in a water bath. The precipitate formed was taken with a spatula, by
dissolving in water. Both sediment and filtrate then tested with Biuret reagent.
Protein precipitation by alcohol. 3 test tubes were prepared. 2ml of albumin
solution with 1 ml of 95% ethanol, 2 ml of albumin solution with 2ml of 95%
ethanol, and 2 ml of albumin solution with 3 ml of 95% ethanol were mixed in
each test tube. The mixture was mixed well and left for 2 minutes. All the test
tubes were observed if they are having insoluble protein.
Protein denaturation. 3 test tubes were prepared, then test tube 1 was mixed with
1 ml of albumin solution and 1 ml of 0.1 M HCl, test tube 2 was mixed 1 ml
albumin solution and 1 ml of 0.1 M NaOH solution, test tube 3 was mixed 1 ml
albumin solution and 1 ml of acetate buffer solution pH 4.7. All the mixtures
mixed well and observed.

RESULTS AND DISCUSSION

The function of Biuret reagent in the experiment is to test the presence of protein.
Millon reagent was not used in the experiment is because Millon reagent is only to
test the presence of tyrosin.
Protein study is very essential and has many applications in veterinary medicine.
An example is veterinary diagnostics in animal disease pathogenesis. Advanced
electrophoresis and mass spectrometry techniques along with recent progresses in

genomics, culminating in bovine and pig genome sequencing. This has widened
the potential application of proteomics in veterinary medicine field (Ceciliani,
2013). Proteomics is the study of structures, functions, and interaction of protein.
Table 1 Results of protein precipitation by metal
Solution
AgNo3

HgCl3

PbAcetate

Picture

esult
+++

++

Explanation : +++ Very cloudy

++ Cloudy
+ Slightly cloudy
From the experiment, all the samples gave out positive results by turning cloudy.
Turning cloudy indicates protein denaturation. The metal ions present in the heavy
metals will break the salt bridge that present in protein resulting proteinat metal
complex. The complex will make protein precipitation. The higher the degree of
ionization of the metal ion, the more the protein will precipitate because more
protein will be denatured ( Wilson & Walker, 2000). In the experiment, the sample
that has most denatured protein was AgNo3, followed by HgCl3 and Pb-Acetate.
The result followed to the literature, because the more reactive the metal, the more
the protein will be denatured. According to the periodic table, the reactivity of
metal is Ag>Hg>Pb.

Table 2 Results of protein precipitation by salt

Solution

Picture

esult
Albumin
(Precipit
ate)

Albumin
(Filtrate)

Explanation : +
-

Contains protein
Does not contain protein

From the experiment, both precipitate and filtrate of protein albumin produced
negative results when tested with biuret reagent. This result did not follow
literature. According to the literature, albumin precipitate should show positive
result which indicates that protein present in precipitate. This is because when
high amount of anorganic salt such as (NH4)2SO4 is added to a protein it will
precipitate by reducing the solubility level of the protein. Protein and salt
molecules both will compete to absorb water molecules ( Bintang, 2010). The
reason why the result did not follow the literature could be because the reagent
used was not in good condition or there have been mistakes that occurred during
experiment. There are two types of protein precipitation by salt salting in and
saltind out. Salting in occurs when solubility of protein increase when salt added.
While salting out occurs when solubility of protein decrease when salt been added
( Yatno, 2008).

Table 3 Results of coagulation test

Solution

Picture

esult
Albumin
(Precipit
ate)

+
Albumin
(Filtrate)

Explanation : +
-

Contains protein
Does not contain protein

From the results, it can be seen that albumin filtrate produced positive result while
albumin precipitate produced negative result when tested with biuret reagent.
According to the literature actually, albumin precipitate should produce positive
result which indicates that protein present in the precipitate. But in this result it
shows that the protein present in the filtrate, this could be because the heat did not
affect the protein to be precipitated fully. When a protein is heated, it will increase
the energy in the solution and change the conformation of the protein. This will
expose the hydrophobic side to the water. Interaction between protein will
increase and causing it to aggregate and finally to precipitate. Protein that has
been denatured will precipitate in isoelectric point. Isoelectric point is a condition
or pH which causes the protein to be in a neutral condition ( Bintang, 2010).

Table 4 Results of protein precipitation by alcohol

Test
Tube
1

Picture

esult
-

Explanation : + Cloudy
- Colourless
From the experiment, only test tube 2 produced positive result by turning cloudy.
Test tube 1 and test tube 2 produced negative result by not changing the colour of
the solution. Turning cloudy in this experiment indicates protein denaturation.
This happenned because alcohol has lower dielectric constant. Dielectric constant
means the ability of the solvent to dissolve the solute. Thus when it is added to the
sample which is albumin in this case the dielectric constant of the sample will be
reduced, resulting in percepitation of the protein. The reason why only test tube 2
has positive result could be because all albumin have reacted with all the ethanol
in the test tube because the ratio of alcohol and albumin was 1:1.

Table 5 Results of protein denaturation ( pH effect).

Solution
HCl

Buffer
Acetate
(pH 4.7)

Picture

esult
-

NaOH

+
+

Explanation : ++ Cloudy
+ Slightly cloudy
From the experiment, test tube 2 and 3 showed postive result by turning cloudy
indicating protein denaturation. Denaturation which caused by pH is irreversible.
This condition is also caused by isoelectric point of a protein. The results did not
follow literature. Actually, according to literature, the protein sample that has been
added by acetate buffer should have produced the most precipitation. This is
because the pH of the buffer is does not differ much from isoelectric point of the
albumin protein. According to Kusumaningrum (2014) isoelectric point of
albumin is pH of 4.6. Adding of HCl should have caused a bit of precipiation and
none when NaoH added. This is because adding of strong acid or base will cause

the protein will be in extreme pH or condition. This will cause denaturation of

protein and protein activity will reduce.

Refrence

Bintang M. 2010. Biokimia Teknik Penelitian. Jakarta (ID) : Erlangga.

Cecialanii (2013) Protemics in Veterinary Medicine.
VeterinaryPathalogyOnlineFirst
Indradjaja A, Suparyatha IB, Hartawan INB. 2014. Hubungan antara kadar
albumin dan mortalitas pasien di unit perawatan intensif anak RSUP Sanglah
Denpasar.Medicina. 45(1): 13-18.
Mcurry J. 2008.Organic Chemistry 8th Edition. New York (US): W.H. Freeman
and Company.
Wilson K, Walker J. 2000. Principles and Techniques of Practical Biochemistry 5 th
Edition. Cambridge (AU): Cambridge University Press.
Yatno, Ramli N, Hardjosworo P, Setiyono A, Purwadaria T. 2008. Sifat kimia dan
nilai biologis konsentrat protein bungkil inti sawit hasil ekstraksi kombinasi fisikkimiawi.Media Peternakan. 31(3): 178-185