(Mæhre, 2018)

BIOCHEMISTRY

BIO462

LABORATORY PRACTICAL

EXPERIMENT 2: PROTEIN DETERMINATION

LECTURER: DR. NUR MAISARAH SARIZAN

GROUP: RAS2012B

GROUP MEMBERS:

NO. NAME STUDENT ID

1. ADDINA BINTI AHMAD HAZRIZAL 2023680198

2. NUR AMIRA FATINI BINTI AHMAD FATHILLAH 2023696428

3. NUR HANIS BINTI SHUHAIMI 2023415484

4. NUR SYAHIRA IZWANI BINTI FAUZI 2023239906

5. NURUL NAJWA BINTI NAZRI 2023423622

INTRODUCTION

Proteins have a major role in the growth and maintenance of the human body along with
carbohydrates and lipids which are the energy giving nutrients in the diet (Mæhre, 2018). In
addition to providing structural support, proteins also pose a wide range of other functions in
the body, such as enzymatic activity, transport of nutrients, hormones, building blocks,
biochemical catalysts, and agents of cellular death. There are 20 common amino acids in
biological chemistry, and these amino acids make up proteins, which are biopolymeric
structures. Four structural levels of proteins which are primary, secondary, tertiary, and
quaternary help to allow further definitions of proteins (Andrew LaPelusa, 2022).

Each of the chemicals have the ability to absorb, transmit, or reflect light within a
certain wavelength spectrum. A spectrophotometer is a device used to quantify the amount
of light a sample can absorb by shining a light beam through a sample and determining the
amount of light reaches the detector. The component of a spectrophotometer consists of a
light source, digital display, sample compartment, monochromator, a wavelength sector to
transmit a selected wavelength, collimator for a straight light beam transmission and a
photoelectric detector. There are two categories into which light sources may be divided
based on their range of wavelengths (Vo, n.d.):

 UV-visible spectrophotometer: utilizes light in the visible (400–700 nm) and ultraviolet
(185–400 nm) regions of the electromagnetic radiation spectrum.
 IR spectrophotometer: utilizes light that falls between 700 and 15000 nm in the
infrared region of the electromagnetic spectrum.

Spectrophotometry is based on The Beer-Lambert Law, which also referred to as Beer's

Law, describes that the absorbance and sample concentration have a linear relationship (Vo,
n.d.). Therefore, using spectroscopy analytically for determining concentrations is based on
the Beer's Law. The methods used in this experiment for protein determination are the
Biuret, Bradford, and Lowry methods. In the Biuret technique, copper interacts with protein
peptide bonds to generate a complex under an alkaline condition. When the Coomassie
Brilliant Blue G-250 dye attaches to an acid-denaturing protein, the Bradford method creates
a vibrant blue color. The Lowry technique is a modification of the Biuret method, which is
more sensitive and somewhat reliant on the composition of amino acids.

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OBJECTIVES

1. To determine the absorbance of the sample using a spectrophotometer.

2. To plot the graph of concentration of BSA against absorbance of samples.
3. To analyze all methods according to sensitivity and convenience.

MATERIALS

BIURET METHOD
 Spectrophotometer
 Calibration cuvette
 Distilled water
 NaKC4H4O6 – 4H2O (9.0 g)
 CuSO4 – 5H2O (3.0 g)
 KI (5.0 g)
 NaOH (0.2 M) (1000 mL)

BRADFORD METHOD
 Spectrophotometer
 Calibration cuvette
 Distilled water
 Bradford reagent

LOWRY METHOD


PROCEDURE

BIURET METHOD
Reagent Preparation

1. The sodium tartrate has been placed into a 1 litre volumetric flask.
2. The sodium tartrate has been dissolved in about 400 mL of 0.2 M NaOH.
3. Then, the copper sulphate and potassium iodide has been dissolved.
4. To a final volume of 9.0 litre has been diluted with 0.2 M NaOH.
5. In a plastic bottle it has been stored.
6. The reagent remained stable indefinitely.

Procedure [Protein range 2.0 mg/mL to 8.0 mg/mL]

1. 1.5 ml of the Biuret reagent has been added to each test tube/

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 2. 1.0 ml of the unknown or protein standard has been added to the test tube.
3. For 30 minutes the test tube has been mixed and incubated at room temperature.
4. OD has been read at 555nm.

BRADFORD METHOD
Reagent Preparation

1. In 50 mL of 95% ethanol, an amount of 100 mg of Coomassie Brilliant Blue G-250

are dissolved.
2. 100 mL of H₃PO₄ (Phosphoric acid) are carefully added and mixed.
3. A final volume of 1000 mL are diluted with water.
4. Bradford reagent are filtered.
5. The reagent are sat until the time to settle and the amount of reagent needed are
carefully decant for analysis.

Procedure [Protein range 0.10 mg/mL to 2.00 mg/mL]

1. 3.0 ml of the Bradford reagent is added to each test tube.

2. 0.06 ml of the unknown solution or protein standard are added to each test tube.
3. At room temperature, each test tube are mixed and incubated for 5 minutes.
4. At 595 nm, OD are readied.

LOWRY METHOD
Reagent Preparation

Procedure [Protein range 0.1 mg/mL to 0.4 mg/mL]

RESULTS

i. Biuret Method

OD OD OD
Concentration
Mean ± S.D
of BSA
(replicate 1) (replicate 2) (replicate 3)

2 mg/ml 0.082 0.084 0.082 0.083 ±

4 mg/ml 0.0133 0.133 0.134 0.133 ±

6 mg/ml 0.165 0.166 0.165 0.165 ±

8 mg/ml 0.188 0.189 0.189 0.189 ±

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 Unknown 0.206 0.206 0.206 0.206 (no S.D)

Blank 0

ii. Bradford Method

Concentration OD OD OD
of BSA (replicate 1) (replicate 2) (replicate 3) Mean ± S.D

0.2 mg/mL 0.723 0.720 0.719 0.721 ±

0.4 mg/mL 0.771 0.771 0.769 0.770 ±
0.6 mg/mL 0.856 0.855 0.853 0.855 ±
0.8 mg/mL 0.898 0.897 0.896 0.897 ±
Unknown 1.225 1.225 1.224 1.225 ±

iii. Lowry Method

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GRAPH

i. Biuret Method (std tak masuk lagi)

0.2
f(x)==0.93860894251242
R² 0.023 x + 0.022
0.18

0.16

0.14

0.12
0D(555nm), A

0.1

0.08

0.06

0.04

0.02

0
0 1 2 3 4 5 6 7 8 9

[Protein Std], (mg/ml)

DISCUSSION

Biuret Bradford Lowry

Convenience Convenient but
involves a 30-minute
incubation period
Sensitivity The least sensitive
method compared to
the Bradford and
Lowry methods can
detect protein
concentrations of up to
0.1 mg with high
accuracy and
independence from
amino acid
composition.
Simple and Require a lot of
timesaving, with only a Steps and the
five-minute incubation incubation time
40 minutes.
High sensitivity It is highly sensitive
depends on the amino to low protein
acid composition. It content and
can detect protein capable of
concentrations of up identifying protein
to 0-0.1 mg. levels of up to 0-0.1
Sensitivity to mg. It is more
interference can lead delicate than the
to errors. It is more Biuret method but
sensitive than the not as sensitive as
Lowry and Biuret the Bradford
methods. method. Its
sensitivity also

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 depends, in part, on
the amino acid
composition.
Generality Consistence Low consistent Pretty consistent
Linearity Linear Linear Linear

Protein concentrations are determined using a variety of factors, including the

amount of protein, the type of protein to be analysed, the presence of substances that
interfere with the measurement, the sensitivity of the apparatus, and the technique employed
(Mæhre, 2018). The protein concentration in BSA was determined using a
spectrophotometer. The three protein assay techniques used were Biuret, Bradford, and
Lowry. A spectrophotometer measures the quantity of light entering a sample and the
amount of light leaving the detector. The instrument displays the absorbance measurement
rather than the transmittance. Absorbance readings are recorded according to the table in
the results section.

Plotting the absorbance versus BSA concentration graphs for each of the three ways
was done using the experiment results. The biuret method, which was the first, involved a
sample with an unknown concentration and concentrations of 2, 4, 6, and 8 mg/mL. The
absorbance values are ____________, in that sequence. The unknown concentration
produces a result of _____. The BSA concentrations for the Bradford method are 0.2, 0.4,
0.6, and 0.8 mg/mL, and the sample is unknown. The readings for absorbance are, in order,
_______The sample's unknown concentration is ___. The BSA values in the Lowry method
include 0.1, 0.2, 0.3, and 0.4 mg/mL, as well as unknown sample. The readings for
absorbances are, in order, _______. the unknown amount of unknown ______. The reading
was taken three times to achieve an average reading to receive an accurate result.

The experiment's errors might be classified as random or systematic, depending on

the contributing causes. Improper spectrometer calibration may have contributed to
systematic inaccuracies. The spectrophotometer can considerably to minimize internal error,
reset the experiment to zero using a blank cuvette. Furthermore, human error might cause
random errors. For improperly cleaning the test tube or using an inaccurate volume of
solution might result in insufficiencies and dilute the solution from its intended consistency.

CONCLUSION

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The objective for this experiment was achieved. The Biuret method is the most appropriate
choice for determining protein content due to its convenience and suitability, especially
considering that the concentrations of unknowns fall within the standard protein range. The
protein concentration for unknown of Biuret method is _____mg/ml while Bradford method is
_____ mg/ml and Lowry method is _____ mg/ml

REFERENCES

Andrew LaPelusa, R. K. (14 November, 2022). Physiology, Proteins. Retrieved from National
Library of Medicine:
https://www.ncbi.nlm.nih.gov/books/NBK555990/#:~:text=Proteins%20serve%20as
%20structural%20support,secondary%2C%20tertiary%2C%20and%20quaternary

Mæhre, H. K. (1 January, 2018). Protein Determination—Method Matters. Retrieved from

National Library of Medicine:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789268/

Vo, K. (n.d.). 2.1.5: Spectrophotometry. Retrieved from LibreTexts Chemistry:

https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbo
ok_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Kinetics/
02%3A_Reaction_Rates/2.01%3A_Experimental_Determination_of_Kinetics/
2.1.05%3A_Spectrophotometry#:~: