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BIOCHEMISTRY MANUAL

Prepared by Admer Rey C. Dablio, v2012


Revised by Mary-Ann A. Landiao, v2016

Experiment 1
Score:

Isolation and Purification of Proteins


Name: Joann H. Justiniane
Groupmates: Marla C. Basa
Ana Margarita L.
Baytion

Date Performed: June 28,


2016
Instructors Signature:

Objectives:

II

To know the isoelectric isolation and purification of protein.


To study the principle used to isolate Albumin from other proteins.
Chemicals:

III

10% hydrochloric acid


1N Acetic acid
1% Silver nitrate

Buffered Ammonium sulfate


Nitric acid
95% Alcohol

Milk
2 eggs
6 Test tubes
Funnel
Funnel Stand
Tong

Apparatus/Materials/Equipment:

25 mL Graduated
Cylinder
Pipette
Litmus paper
Stirring rod
250 mL Beaker
Watch glass

IV

Schematic Diagram of the Procedure:


Please write on a short bond paper.

Summary of Theory

Milk is the most nutritionally-complete food that can be found in nature. Milk
contains vitamins (principally thiamine, riboflavin, pantothenic acid, and vitamins A,
D, and K), minerals (calcium, potassium, sodium, phosphorus, and trace metals),
proteins (which include all the essential amino acids), carbohydrates (chiefly lactose),
and lipids (fats). There are three kinds of proteins in milk: caseins, lactalbumins, and
lactoglobulins. Casein is the main protein found in milk. It has a phosphate group
attached to the hydroxyl group of some of the amino acid side chains. Casein exists
in milk as the calcium salt called calcium caseinate.
Calcium caseinate has an isoelectric point of pH 4.6 and is generally the pH at
which the protein is least soluble. The pH of milk is about 6.6; therefore, casein has a
negative charge at this pH and is solubilized as a salt.
Casein, as a curd protein, consists of many components bound up together
into bundles called micelles. A protein subunit stabilizes each of the micelles to
keep them dispersed into separate micelles throughout the milk. Upon acidification,
the protective protein subunit is disturbed and the micelles can clot together, which
No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

then makes the milk curdle or coagulate. Chemically, the casein micelle is damaged
and therefore, aggregated due to the decrease of its isoelectric charge. At that same
time, the acidity of the medium increases the solubility of minerals
so that organic calcium and phosphorus contained in the micelle gradually become
soluble in the aqueous phase. Casein micelles disintegrate and casein precipitates,
resulting in clumps.
When washing the curd, some of the fats were removed due to the addition of
alcohol. Alcohol was used to remove fats because alcohol can dissolve both oilsoluble and water-soluble compounds.
Albumin are a family of globular proteins. They are water-soluble and
commonly found in blood plasma. Types include serum albumin and storage protein
ovalbumin found in egg whites.
Addition of Ammonium sulfate pulls water molecules away from the non-polar
units of proteins. The decrease in available water molecules increases the surface
tension and enhances hydrophobic interactions, thus allowing the protein to
precipitate from a solution or bind to a hydrophobic column. The use of ammonium
sulfate has the following advantages: 1) High concentrations of ammonium sulfate
inhibit microbial growth and maintain the protein in a folded state. 2) The low density
of saturated solutions (1.25 g/cm3) allows pelleting of proteins by centrifugation. 3) A
low heat of solubilization avoids the risk of protein denaturation that can occur when
the sample temperature increases.
The supernate was discarded because it does not contain the Albumin.
VI

Observations

Table VI.1 Isolation of Casein from Milk


Brand of Milk
Volume of Milk Used, mL
# of 10% HCl Drops Added
Weight of Watch glass + filter paper
# of Washings done before filtrate becomes Cl- free
# of HNO3 drops added
Weight of Watch glass + filter paper + Residue
Odor of the Residue
Description of Precipitate after air drying
Weight of Protein Isolated
Casien Yield (g/mL)
Table VI.2 Isolation of Egg Albumin
Volume of Egg White Used, mL
Volume of (NH4)2SO4, mL
Describe mixture after 30 minutes
Describe the precipitate after centrifugation
Describe the mixture after 2 days
Description of Precipitate
Weight of Protein Isolated
Albumin Yield (g/mL)

Alaska
12.5 mL
12
33.97 g
1
2
35.10 g
Rancid odor
White powder
1.13 g
0.0904 g/mL

50 mL
50 mL
Appearance of 3 layers
Slightly yellowish precipitate
White gob
White powder
2.9383 g
0.0588 g/mL

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

Data

Table a.1 1st Weighing (Beaker + Test Tube)


Test Tube
1st Reading
2nd Reading (g)
(g)
1
12.8039
12.8040
2
12.6908
12.6907
3
12.9572
12.9573
4
12.7683
12.7684
5
12.8952
12.8953
6
12.8205
12.8205
Table a.2 2nd Weighing (Beaker + Test Tube)
Test Tube
1st Reading
2nd Reading
(g)
(g)
1
12.8040
12.8042
2
12.6908
12.6909
3
12.9575
12.9876
4
12.7684
12.7688
5
12.8952
12.8953
6
12.8208
12.8209
Table a.3 Residue
Test
1st Weighing
Tube
(g)
1
12.8040
2
12.6907
3
12.9572
4
12.7684
5
12.8953
6
12.8205

2nd Weighing
(g)
12.8040
12.6910
12.9576
12.7688
12.8953
12.8209

3rd Reading
(g)
12.8041
12.6906
12.9572
12.7684
12.8953
12.8205

3rd Reading
(g)
12.8043
12.6912
12.9577
12.7687
12.8954
12.8209

Average (g)
12.8040
12.6907
12.9572
12.7684
12.8953
12.8205

Average (g)

Beaker +
Test Tube + Residue
12.8377
12.7726
12.5584
13.1896
13.2663
14.2509

TOTAL

12.8040
12.6910
12.9576
12.7688
12.8953
12.8209

Residue
0.0337
0.0816
0.6012
0.4210
0.3710
1.430

2.9383 g

RESIDUE :
VII

Analysis

Protein purification includes a series of processes to isolate a particular


protein from a complex mixture. This technique is critical for characterizing the
function, structure, and interaction of the protein of interest. An analytical purification
generally uses three distinct properties to separate proteins, including the isoelectric
technique.
A protein can be purified according to its protein isoelectric point by running
the said protein through an ion exchange column or a pH-graded gel. At the

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

isoelectric point, a protein has no net charge. Above the isoelectric point, a protein
carries a net negative charge, below it is a net positive charge.
Because a majority of weakly acid remains in nearly every protein, they are
generally negatively charged at neutral pH. The isoelectric point is significant in
protein purification because it represents the pH where solubility is typically minimal.
Here, the protein isoelectric point signifies where mobility in an electro-focusing
system is zero and, in turn, the point where the protein will collect.
To purify proteins using the isoelectric focusing method, we added 12 drops of
10% HCl to the milk. Upon addition of acid, the protein molecules simultaneously
migrated in the solution until the milk reached their protein isoelectric point which is
4.6. In testing for the presence of chloride ion in the milk, we added 1% AgNO 3 to the
filtrate. To avoid losing too much casein, we washed the filtrate with dilute HNO 3.
The constant weight was obtained by calculating the averages of the
successive measurements. The difference of 0.0006 g is considered a constant
weight since these successive weighing have high precision and will be near to the
true value. The constant weighing process plays a significant role in producing data
with less error since the initially absorbed excess water in the test tubes and the
samples and the moisture that the samples can acquire when exposed to air can add
to the net calculated yield of albumin.
VIII

Conclusion

In this experiment, casein was precipitated because the phosphate groups are
protonated by adding 10% HCl. After adding the acid, the pH was lowered from 6.6 to
4.6, this affects the solubility of the milk protein casein forming curds.
The spoilage of milk was due to sudden change of temperature. When this
occur, a bacteria such as Lactococci and Lactobacilli is formed. These bacteria will act
on the lactose present in milk to produce the sour Lactic acid. They hydrolyzes
lactose only from the galactose portion of the lactose. Since the production of the
lactic acid also lowers the pH of the milk, the milk clots when it sours:

This is the reaction process in producing Lactic Acid that is responsible for souring the
milk.
Freezing and filtration was the process used to extract Albumin. This breaks
the tissue or cell and makes the soluble protein to separate from the insoluble type.
The albumin can be isolated from other proteins by centrifugation.
The purification of albumin was not accurate due to equipment limitations and
observation errors. The students should properly study the procedure and research
some techniques used in the experiment.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

IX

References
Wilkins TD, Velander W. J Cell Biochem. 1992 Aug;49(4):333-8
Jeremy M Berg, John L Tymoczko, and Lubert Stryer. Biochemistry 5th Edition
2002.
McMaster University - Chem2O06 Lab Manual (taken July 2, 2016)
Paul Messier, Topics in Photographic Preservation. Vol. 4, 1991. pp. 124-135
https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Bulletin/a5479bul.pdf (taken July 2, 2016)
http://info.gbiosciences.com/blog/bid/149959/What-is-the-Role-of-theIsoelectric-Point-of-a-Protein-in-its-Purification (taken July 2, 2016)
http://www.isonovatech.com/egg-protein-isolate/ (taken July 2, 2016)
Factors affecting the pH-Sensitivity of the Heat Stability of Milk from Individual
Cows, August 1961. Vol. 44, Issue 8, Page 1405-1413
http://www.food-info.net/uk/qa/qa-fp27.htm (taken July 2, 2016)

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

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