STRUCTURAL BIOCHEMISTRY (UDEE 1204)
BACHELOR OF SCIENCE (HONS) BIOCHEMISTRY
YEAR 1 SEMESTER 2
SESSION: MARCH 2019
DATE: 7 MARCH 2019
LECTURER: DR. CHAI TSUN THAI
EXPERIMENT 5
TITLE: MEASUREMENT OF PROTEIN SOLUTIONS
STUDENT’S NAME:
NG YI QING
STUDENT’S ID:
1806700
LAB PARTNERS:
1. LOW SHEAU TONG 1706117
2. CHEAH HUI LE 1703931
3. YONG CHIA XIN 1703855
Title:
Measurement of Protein Solutions
Objective:
1. To learn the measurement of protein solutions.
2. To investigate the difference between color change of peptide bond and Biuret
compound.
Introduction:
Proteins are large biomolecules, or macromolecules, consisting of one or more long
chains of amino acid residues. Proteins perform a vast array of functions within
organisms, including catalysing metabolic reactions, DNA replication, responding to
stimuli, providing structure to cells and organisms, and transporting molecules from
one location to another. Protein is also a macronutrient that is essential to building
muscle mass (Szalay,J.2019).Proteins in particular are a biochemical compound that
need to be measured oftenly. Due to the presence of tyrosine and tryptophan, proteins
and peptides containing these aromatic amino acids absorb UV light at a wavelength
of 280 nm (Protein Man,T.2019). Most pure protein solutions containing 1 mg/mL of
protein have an absorbance of about 1.0 when the light path is 1 cm. This method is
simple, rapid, and allows for full recovery of the protein. However, an accurate
quantitation will be difficult due to many other biochemical absorb near this
wavelength. Furthermore, the absorbance of different proteins will be different extents
depending on their aromatic amino acid. The biuret test, also known as Piotrowski's
test, is a chemical test used for detecting the presence of peptide bonds and avoiding
the problem above. It is made of sodium hydroxide and copper sulphate. In the
presence of peptides, a copper(II) ion forms mauve-colored coordination complexes
in an alkaline solution.
Material & Method:
Spectrophotometer, test tubes, pipettes, vortex, Bovine serum albumin (BSA), Biuret
reagent
A stock solution of bovine serum albumin (10mg/mL) is being used, serial dilution of
2,4,6,8, and 10mg/mL is prepared. (Final volume for each tune is 1mL)
↓
9mL Biuret reagent is added to each tube, it is vortexed immediately, and is let stand
at room temperature for 20 minutes.
↓
1mL of the sample labelled UNKNOWN is pipetted to a test tube and step 3 is
repeated.
↓
The absorbance is read at 550nm and spectrophotometer is being used.
↓
A graph of absorbance versus concentration is plotted and the concentrations of the
UNKNOWN samples are determined from the curve.
Results:
Bovine serum
Volume of
Volume of
Volume of
Absorbance of
albumin
BSA stock
water (μL)
Biuret reagent
550nm
(mg/mL)
(μL)
0
0
1000
2
200
800
↑
0.165
4
400
600
9000
0.229
6
600
400
↓
0.289
8
800
200
0.336
10
1000
0
0.371
Unknown
○,1Biuret reagent provided
○,21000μL sample
(μL)
0.000
0.231
+
9000μL Biuret
↓
Mix
↓
Stand 15 min
↓
OD550nm
○,3Bovine serum albumin
(BSA) → 1%
= 1/1000mLg
= 1000mg/100mL
= 10mg/mL
M1V1 = M2V2
10mg/mL x V1 = 2mg/mL x 1000μL
V1 = (2 x 1000) / 10
= 200μL
M1V1 = M2V2
For 0mg/mL,
10mg/mL x V1 = 0mg/mL x 1000μL
V1 = (0 x 1000) / 10
= 0μL
For 2mg/mL,
10mg/mL x V1 = 2mg/mL x 1000μL
V1 = (2 x 1000) / 10
= 200μL
For 4mg/mL,
10mg/mL x V1 = 4mg/mL x 1000μL
V1 = (4 x 1000) / 10
= 400μL
For 6mg/mL,
10mg/mL x V1 = 6mg/mL x 1000μL
V1 = (6 x 1000) / 10
= 600μL
For 8mg/mL,
10mg/mL x V1 = 8mg/mL x 1000μL
V1 = (8 x 1000) / 10
= 800μL
For 10mg/mL,
10mg/mL x V1 = 10mg/mL x 1000μL
V1 = (10 x 1000) / 10
= 1000μL
Absorbance at 550nm
0,45
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
4,98; 0,231
0
2
4
6
8
Bovine serum albumin (mg/mL)
10
12
Discussion:
The presence of amino and carboxylic acid groups enables amino acids to accept
protons from and donate protons to aqueous solution, and, therefore, to act as acids
and bases. Because proteins contain both acidic and basic side-chains, they too can
donate or accept protons. A molecule that functions simultaneously as an acid and a
base is known as an amphoteric molecule. The biuret test for proteins positively
identifies the presence of proteins in solution with a deep violet color. Biuret,
H2NCONHCONH2, reacts with copper (II) ions in a basic solution to form a deep
violet complex (Kumar,P.2019). From the experiment above, we have read the
absorbance at 550nm using different bovine serum concentration. 10mg/mL of bovine
serum albumin and distilled water is used to create different concentration of sample.
The Biuret reagent solution is let to stand for 15 minutes after dilution. From the
results obtained from spectrophotometer, more proteins will be absorbed indicates
greater absorbance due to the higher concentration of the sample. The graph plotted
shows the absorbance at 550nm is slightly directly proportional to the bovine serum
albumin concentration. Furthermore, the reading of spectrophotometer at OD550 for
unknown bovine serum that showed from the graph is 0.231, thus the concentration of
unknown bovine serum albumin is 4.98mg/mL.
Conclusion:
In conclusion, different concentration of sample will lead to different absorbance of
550nm. The graph of absorbance versus concentration is plotted according to the table.
The graph is slightly directly proportional to the bovine serum albumin concentration.
The reading of spectrophotometer at OD550 for unknown bovine serum that showed
from the graph is 0.231 and the concentration is 4.98mg/mL. In accordance, the range
concentration of unknown bovine serum is between 4-6mg/mL.
References:
1. Szalay, J. (2019). What Is Protein?. [online] Live Science. Available at:
https://www.livescience.com/53044-protein.html [Accessed 10 Dec. 2015].
2. Protein Man, T. (2019). Why Does Tyrosine and Tryptophan Have Effect in
Protein Determination and to What Degree?. [online] Info.gbiosciences.com.
Available at:
https://info.gbiosciences.com/blog/why-does-tyrosine-and-tryptophan-have-effectin-protein-determination-and-to-what-degree [Accessed 17 Feb. 2016].
3. Kumar, P. (2019). Qualitative and Quantitative Tests for Amino Acids and
Proteins. [online] Biology Discussion. Available at:
http://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-foramino-acids-and-proteins/13065 [Accessed 9 Mar. 2019].