SBL 1023

TECHNIQUE IN BIOLOGY AND

BIOCHEMISTRY LABORATORY

LAB 4: PROTEIN ANALYSIS

STUDENT NAME MATRIC NUMBER LECTURE GROUP

ALPHABET

DIVYARSI A/P E20182022971 E

THELLAIVASSAN

LINGESSWARI A/P E20182022985 E

NAGARAJAN

PRAMILLA A/P E20182022989 E

PERIASAMY

CHAAVENEA A/P E20182022973 E

POOBALAN
AIM/ OBJECTIVE:

The qualitative method used which is by using biuret reagent. It was able to
show the presence of proteins in the albumin, Soy bean and fish oil by detecting
the presence of peptide bonds.
When the peptide bonds present in an alkaline solution, the coordination complexes
Associated with a copper ion (Cu2+)are violet in colour.Nitrogen atoms of the peptide
bonds form a coordination bond with metal ion.The quantity of the complexes formed
is proportional to the number of peptide bonds.Thus, protein intensity affects the
intensity of the colour,where the colour will be more intense with more protein.

MATERIALS :

 Stock solution of Bovine Serum Albumin(BSA):10mg/ml

 Deionized water(dH2O)
 Test tubes and stand
 Pipette
 Biuret reagent
 Spectrophotometer
 Protein samples
METHODOLOGY :
(A) Preparation of biuret reagent
Add, with stirring,300ml of 10%(w/v) NaOH to500ml of a solution containing 0.3%
Copper sulfate pentahydrate and 1.2%sodium potassium tartarate, then dilute to 1
liter.

(B) Protein preparation

1.Prepare 2 sets of test tubes with the numbers 1 to 6 and prepare the bovine serum
albumin.
(BSA) stock solution(10mg/ml)according to the concentration listed below:

2. Prepare duplicate test tubes for protein samples and carefully pipette 1ml of the
protein samples in to each tubes.
3. Add 2ml of biuret reagent to every tube:the 14 tubes for the standard curve and the
duplicate tubes for protein samples.
4. Cover the tubes with parafilm and briefly vortex to ensure that the protein
standards/samples and the biuret reagent are thoroughly mixed.
5. Allow the tubes to stand at 15minutes.
6. Switch on the spectrophotometer and adjust the wavelength to 550nm.
(C) Determine protein concentration
1. Transfer 1ml of solution from tube 1 into a cuvette and gently wipe the cuvette
with a paper towel to remove fingerprints and dust.
2. Set the absorbance to 'zero'.This tube will serve as 'blank'.
3. Measure the absorbance of the other standards and sample proteins using the
stepasin(1).

*DO NOT blank the instrument again.

4. Record the absorbance of each standards and samples.
5. Plot a graph of standard curve using the absorbance value of protein standards
and interpolate the absorbance values of the protein samples.
RESULT ANALYSIS

Test tubes Wavelengths

A (Albumin) 0.708

B (Soy bean) 2.600

C (fish) 0.395

Test tubes BSA stock(mL) H2O(mL) Wavelengths

D 0.9 0.1 0.103

E 0.8 0.2 0.191

F 0.7 0.3 0.323

G 0.6 0.4 0.380

H 0.5 0.5 0.436

I 0.4 0.6 0.580

GRAPH ANALYSIS

The biuret protein assay was published as a method to determine protein

concentration in the 1940s, although the reaction itself was studied as long ago as the
early 19th century. The biuret assay was commonly employed well into the 1980s and
is still in use because it is so convenient and inexpensive to prepare and easy to use.
When we assay a protein sample we lose some of it because the colorimetric reagent
destroys the protein in the process. Because the biuret assay consumes a lot of protein
many laboratories use methods that employ a much more sensitive color reagent such
as the Bradford assay. We use the biuret assay for this laboratory study because you
can prepare your own reagent from inorganic reagents (i.e., from "scratch"), giving
you an opportunity to practice your solution-making skills.

Wavelengths vs Standard solutions

0.7
0.6
0.5
Wavelengths

0.4
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7
Standard solutions D to J

Wavelengths VS Protein samples

3

2.5
Wavelengths

1.5

0.5

0
0 0.5 1 1.5 2 2.5 3 3.5

Protein samples
DISCUSSION:
The biuret reagent is not named after z famous scientist but after a substance
called biuret (H2NC(O)NHC(O)NH). Biuret is the result of the condensation of 2
molecules of urea. The reagent is so named because the peptide bonds in biuret give a
positive result for the test.
A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide
bonds under alkaline conditions. The biuret reagent, which contains all the chemicals
required to carry out the analysis, can be purchased commercially. It is mixed with a
protein solution and then allowed to stand for 15-30 minutes before the absorbance is
read at 540 nm. The major advantage of this technique is that there is no interference
from materials that adsorb at lower wavelengths, and the technique is less sensitive to
protein type because it utilizes absorption involving peptide bonds that are common to
all proteins, rather than specific side groups. However, it has a relatively low sensitivity
compared to other UV-visible methods.

OBSERVATION INFERENCES
No change (solution remains blue ) Proteins are not present

The solution turns from blue to violet Proteins are present

(deep purple)
The solution turns from blue to pink Peptides are present (Peptides or
peptones are short chains of amino acid
residues)

The Biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate
complex with peptide bonds (-CONH- groups) in alkaline conditions. Lone electron
pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion to
form the chelate complex. The chelate complex absorbs light at 540 nm so
appears violet. Hence a color change from blue to violet indicates that proteins are
present. The greater the concentration of peptide bonds, the greater the color intensity.
If the concentration of peptide bonds is low – such as when short-chain peptides are
present - the color change is from blue to pink.
Reference:

Protein products

Test tubes of BSA stock