PRACTICAL 3: ANALYSIS OF SOLUBILISED PROTEIN-

BIURET REAGENT METHOD

NAME : AINA KAMILIA BINTI NORAZLAN

MATRIC NO. : 201079

COURSE : BIOPHYSICAL CHEMISTRY (BTC3000)

PROGRAMME : BACHELOR OF SCIENCE IN

BIOTECHNOLOGY

LECTURER : DR FADZLIE WONG BIN FAIZAL WONG

INTRODUCTION
The determination of protein concentration is an essential technique in all aspects of
protein studies and proteomics. Although there are a wide variety of protein assays
available none of the assays can be used without first considering their suitability for
the application. Each method has its own advantages and limitations and often it is
necessary to obtain more than one type of protein assay for research applications.
The dye binding protein assays are based on the binding of protein molecules to
Coomassie dye under acidic conditions. The binding of protein to the dye results in a
spectral shift, the color of Coomassie solution changes from brown (absorbance
maximum 465nm) to blue (absorbance maximum 610nm). The change in color density
is read at 595nm and is proportional to the protein concentration.
In the copper ion based protein assays, protein solutions are mixed with an alkaline
solution of copper salt, cupric ions (Cu2+). The protein assay is based on the
interactionof cupric ions with protein in an alkaline solution and is commonly referred
to as the Biuret assay. The interaction of cupric ions (Cu2+) with protein results in a
purple color that can be read at 545nm. The amount of color produced is proportional
to protein concentration.

Under alkaline conditions cupric ions (Cu2+) chelate with the peptide bonds resulting
inreduction of cupric ions (Cu2+) to cuprous ions (Cu+). The Cuprous ions can also
be detected with Folin Ciocalteu Reagent (phosphomolybdic/phosphotungstic acid);
this method is commonly referred to as the Lowry method. Cuprous ions (Cu+)
reduction of Folin Ciocalteu Reagent produces a blue color that can be read at 650-
750nm. Theamount of color produced is proportional to the amount of peptide bonds,
i.e. size as well as the amount of protein/peptide.

OBJECTIVE
 Learn the principles of protein assays.
 Determine protein concentrations using the Biuret Protein Assay
RESULTS
Table 1: Reading of absorbance 550nm for different amounts of BSA standard solution
Reagent Test
tubes
1 2 3 4 5 6
Amount of BSA (ml) 0 0.1 0.2 0.3 0.4 0.5
Distilled water (ml) 0.5 0.4 0.3 0.2 0.1 0
Biuret reagent (ml) 2.5 2.5 2.5 2.5 2.5 2.5
Amount of BSA 0 2 4 6 8 10
(mg/ml)

Average absorbance 0 0.164 0.210 0.306 0.388 0.454

at 550nm (A)

Table 2: Reading of absorbance 550nm for milk powder solutions

Type of Reading absorbance 550nm powder solutions of
milk milk
powder solution
A B C Average Blank Final
Average reading
Similac 0.400 0.392 0.350 0.381 0.154 0.227
Anmum 0.307 0.291 0.313 0.304 0.154 0.150
Dutch 0.327 0.319 0.312 0.319 0.154 0.165
Lady
Pedia 0.332 0.362 0.345 0.346 0.154 0.202
Sure
 Graph Absorbance (A) against Amount of BSA (mg/ml)
0.6

0.5 y = 0.0484x
R² = 0.9562

0.4
Absorbance (A)

0.3

0.2

0.1

0
0 2 4 6 8 10 12
Amount of BSA (mg/ml)

CALCULATION
1. Calculate the amount of solubilised protein of milk powder solutions that
corresponds to the absorbance.
y = 0.0484x
y = absorbance
x = concentration
Similac 0.227 = 0.0484x
x = 4.69mg/ml
Amount = 4.69mg/ml x 0.5ml
= 2.35mg
Anmum 0.150 = 0.0484x
X = 3.10mg/ml
Amount = 3.10mg/ml x 0.5ml
= 1.55 mg
Dutch 0.165 = 0.0484x
Lady X = 3.40mg/ml
Amount = 3.40mg/ml x 0.5ml
= 1.70mg
Pedia 0.202 = 0.0484x
Sure X = 4.17mg/ml
Amount = 4.17mg/ml x 0.5ml
= 2.09mg
2. Calculate the concentration of solubilised protein in mg/mL milk powder.

y = 0.0484x
y = absorbance
x = concentration

Similac 0.227 = 0.0484x

x = 4.69mg/ml

Anmum 0.150 = 0.0484x

X = 3.10mg/ml

Dutch Lady 0.165 = 0.0484x

X = 3.40mg/ml

Pedia Sure 0.202 = 0.0484x

X = 4.17mg/ml

3. Calculate the amount of solubilised protein containing in 1 g of milk powder.

Each protein:
𝑜. 5𝑔.
=
100𝑚𝑙
0.5𝑔 × 2
1𝑔 =
100𝑚𝑙 × 2
1𝑔
1𝑔 =
200𝑚𝑙
Similac x = 4.69mg/ml
= 4.69mg/ml x 200ml x 10−3
= 0.938mg

Anmum X = 3.10mg/ml
= 3.10mg/ml x 200ml x 10−3
=0.62mg
 Dutch Lady X = 3.40mg/ml
= 3.40mg/ml x 200ml x 10−3
= 0.68mg
Pedia Sure X = 4.17mg/ml
= 4.17mg/ml x 200ml x 10−3
= 0.834mg

QUESTIONS

1. Advantage of Biuret Assay

In alkaline solutions, cupric ion complexes with the peptide bonds of proteins and
peptides to form a purple charge transfer complex (λmax = 540 nm). The intensity of
the color is proportional to the protein concentration. This reaction occurs only with
the peptide bond and not with the amino acid side chains. Because the number of
peptide bonds per given unit weight is approximately the same for all proteins, this
method is generally applicable and reasonably accurate, irrespective of the
composition of the protein mixture. Other advantages of this method are that the
color development time is relatively short and the color intensity remains constant for
a reasonable amount of time (at least 30 minutes). This method also does not
require a spectrophotometer capable of measuring in the UV region. There are
relatively few materials that could interfere with the assay which increases its
accuracy and practicality. This method is considered good for whole tissue samples
and other sources of high protein concentration.

2. Disadvantage of Biuret Assay

A major disadvantage of this assay is its lack of sensitivity, the lower limit being 2 mg
of protein. Greater sensitivity can be achieved by measuring the absorbance of the
protein –cupric ion complex at 310 nm rather than at 540 nm; however, because so
many substances found in crude protein solutions absorb in the nearultraviolet
region, this approach is usually impractical, even when appropriate blanks are
included. Another disadvantage with this assay is that some compounds used in the
laboratory such as Tris buffer and ammonium sulfate, as well as endogenous
compounds in crude extracts, can interfere with color development or generate
colored complexes themselves. These interferences can be minimized by analyzing
protein precipitates. This method could not measure the concentration of proteins
precipitated using ammonium sulfate. This method also requires setting up a
standard curve in order to obtain results. Other than that, nucleic acid contamination
is one of the issues that might arise when carrying out this method. Last but not
least, the disadvantage of this method is that proteins with abnormally high or low
percentage of aromatic amino acids will give high or low readings.

DISCUSSION
The Biuret Assay, also known as the Piotrowski Test, is a biochemical assay
that allows one to accurately quantify protein concentration within the range of 5-150
mg/mL. The protein sample, irrespective of its composition, is measured through
absorbance spectroscopy at 550 nm in conjunction with a known protein concentration
sample. It is also a chemical test used for detecting the presence of peptide bonds
which is suitable to estimate protein concentration between 1 to 20 mg/ml. In the
presence of peptide bond, a copper (ll) ion forms violet-blue colour. Copper (ll) ion
binds with nitrogen atoms present in the peptides of proteins. In a secondary reaction,
the copper (ll) is reduced to copper (l). Buffers such as Tris and ammonia interfere
with this assay. Thus, it will combine the protein samples with Biuret Reagent which
contains copper ions in a basic solution. The copper ions will form a complex ion with
the amide groups in the proteins. Then a blue colour solution will be formed and it will
be measured by using a spectrophotometer. The amount of blue color that forms is
directly proportional to the quantity of protein in the samples. The colour intensity of
the solution increases as the concentration of albumin increases.
To determine the amount of protein quantitively based on the absorbance
reading on the spectrophotometer, a standard curve must be set up beforehand. Due
to Beer-Lambert law, the absorbance reading is directly proportional to the
concentration. High absorbance reading means that there are a lots of molecules
interact with the light. The graph of absorbance against protein concentration was
plotted above. The equation for the graph is y = 0.0484x.
The precaution steps taken when carrying out this experiment are the
absorbance readings on the spectrophotometer was taken 3 times and the average
reading was calculated to obtain an accurate value. Gloves were also used in this
experiment to ensure that fingerprints can be reduced to avoid the inaccuracy of
absorbance reading.

CONCLUSION
In conclusion, biuret assay is one of the ways to quantitatively measure
protein concentration in a solution. From the results obtained, the brand Similac has
the highest protein concentration in its milk solution followed by Pedia Sure, Dutch
Lady and Anmum.
REFERENCES
1. "Biuret Protein Assay." Jenway. Bibby Scientific.
http://www.jenway.com/adminimages/P09_002A_Biuret_Protein_Assay.pdf

2. “Chemistry of Protein Assays | Thermo Fisher Scientific - US.” Thermo Fisher

Scientific, Thermo Fisher Scientific, www.thermofisher.com/us/en/home/life-
science/protein-biology/protein-biology-learning-center/protein-biology-
resource-library/pierce-protein-methods/chemistry-protein-assays.html.

3. Nunez, Jacqueline. “Biuret Assay.” Mycrobe, Mycrobe, 30 July 2018,

https://www.mycrobe.org/blog/2018/7/26/biuret-assay.

4. “Overview of Protein Assays Methods.” Thermo Fisher Scientific - US,

https://www.thermofisher.com/my/en/home/life-science/protein-
biology/protein-biology-learning-center/protein-biology-resource-library/pierce-
protein-methods/overview-protein-assays.html.