DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE AND MATHEMATICS

SULTAN IDRIS EDUCATION UNIVERSITY

SBL 1023
TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY
EXPERIMENT 3: SPECTROPHOTOMETRY (PROTEIN
CONCENTRATION MEASUREMENT)
BY

NAME NUR NATRAH NATASHA BT

MUSTAPA AYUB
MATRIC NUMBER E 20161015941
GROUP B
LECTURE’R NAME PROFESSOR MADYA DR.
SHAKINAZ BT DESA

Date and time of practical class

21th November 2017 2.00 p.m. – 5.00 p.m.

TITLE: SPECTROPHOTOMETRY (PROTEIN ANALYSIS)

INTRODUCTION

Protein can be found throughout our body. Protein is the most plentiful substance after water,
20 per cent of our body consists of protein. Long chain of amino acids which derive from
carbon, oxygen, hydrogen and nitrogen build up the proteins and peptide bonds are
responsible for linking the amino acids.

In this experiment, the way to investigate the concentration of protein in a mushroom

by using a spectrophotometry test and biuret test. So, we decide to observe on 2 sample of
mushroom. There is sample A and sample B. In sample A we used a raw mushroom and
sample B we decide to use a cooked mushroom.

The biuret test is a chemical test used for detecting the presence of peptide bonds. In
the presence of peptides, a copper (II) ion forms violet colour coordination complexes in an
alkaline solution. The Biuret reaction can be used to assess the concentration of proteins
because peptide bonds occur with the same frequency per amino acid in the peptide. The
intensity of the colour and the absorption at 550 nm is directly proportional to the protein
concentration.

A spectrophotometry is employed to measure the amount of light that a sample

absorbs. The instrument operates by passing a beam through a sample and measuring the
intensity of light reach the detector. The beam of light consists of a stream of photons. When
a photon encounters an analyte, the analyte will absorb the photon. This absorption reduce the
number of photon in beam of light.

OBJECTIVE

To determine the protein concentration by using a biuret test and spectrophotometry test in
mushroom.
MATERIALS

Stock solution of Bovive Serum Albumin (BSA): 10 MG/ML

Deionized water (dH20)
Test tube and stand
Pipette
Biuret reagent
Spectrophotometer
Protein sample (Mushroom)

METHODOLOGY

A. Protein preparation
1. One set of test tube with number 1 to 6 and the bovine serum albumin (BSA) stock
solution (10 mg/ml) was prepared according to the concentration listed below:
Test tube BSA concentration H2O (ml) BSA stock (ml)
(mg/ml)
1 0 1.0 0
2 1 0.9 0.1
3 2 0.8 0.2
4 3 0.7 0.3
5 4 0.6 0.4
6 5 0.5 0.5
7 6 0.4 0.6

Table 1: preparation to the concentration

2. Duplicate test tube for protein was prepared and the protein sample was pipette carefully
in each test tube.
3. 2 ml of biuret reagent was added in each test tube, the 7 for the standard curve and the
duplicate tubes for protein samples.
4. The tube was covered by parafilm and vortex to ensure that protein and the biuret reagent
was thoroughly mixed.
5. The tube was allowed to stand for 15 minutes.
6. The protein was measured by spectrophotometer by using the 550 nm of wavelength.
B. Determine protein concentration
1. 1 ml of solution was transfer from tube 1 into a cuvette and gently wipes the cuvette with
a paper towel to remove fingerprint and dust.
2. The absorbance was set to ‘zero’. This tube will serve as ‘blank’.
3. The absorbance of the other standards and the sample protein was measure by using the
step as in 1. *DO NOT blank the instrument again.
4. The absorbance of each standard and sample was recorded.
5. A graph of standard curve using the absorbance value of protein standards and interpolate
the absorbance values of the protein sample was plotted.

RESULT

Diagram 1 Diagram 2

Diagram 1: Sample A and Sample B

Diagram 2: concentration of 0, 1, 2, 3, 4, 5, 6, Sample A and Sample B

Test tube BSA stock H2O (ml) BSA Absorbance Biuret

(ml) concentration (nm) reagent
(mg/ml) (ml)
1 0 1.0 0.00 0.000
2 0.1 0.9 1.00 0.016
3 0.2 0.8 2.00 0.068 Add 2 ml of
4 0.3 0.7 3.00 0.072 biuret
5 0.4 0.6 4.00 0.221 reagent and
 6 0.5 0.5 5.00 0.278 incubate for
7 0.6 0.4 6.00 0.500 15 minutes
8 Sample A 10.0 3.90 0.213
9 Sample B 13.0 0.98 0.015

Table 2: Data of concentration

Standard curve of absorbance value

0.6
0.5
0.5
absorbance (nm)

0.4
Sample B 0.278
0.3 0.221
Absorbance: 0.015 nm
0.2 Con: 0.98 mg/ml
Sample A
0.068 0.072
0.1 Absorbance: 0.213 nm
0 0.016
Con: 3.90 mg/ml
0
0 1 2 3 4 5 6 7
concentration of protein (mg/ml)

Diagram 3: Graph standard curve of absorbance value

CALCULATION

Sample A Sample B
9.99 g of sample = 10.0 g of sample 12.76 g of sample = 13.0 g of sample
+ 10 ml dH2O = 1 ml + 13 ml dH2O = 1 ml
1 g of sample = 3.90 mg/ml concentration of 1 g of sample = 0.98 mg/ml concentration of
protein protein
3.90 mg/ml × 10 ml(dilute sample) = 39.0 0.98 mg/ml × 13 ml(dilute sample) = 12.74
mg mg
3.90 mg × 10−3 = 0.04 g 12.74 mg × 10−3 = 0.01 g
DISCUSSION

In this experiment, the method that used to determine the concentration of protein is biuret
test and spectrophotometry test. From the result, the colour of solution in each test tube
shows the different coloured. Coloured in test tube 7 become more intense than test tube 1.
From the preparation of concentration in Table 1, test tube 7 contains 0.6 ml stock solution of
bovive Serum Albumin (BSA) with 0.4 ml of distilled water and 2 ml of biuret reagent. So,
from this data I can conclude that the higher the concentration of BSA stock solution the
colour of protein become more intense.

From the graph in Diagram 3, the absorbance of the spectrophotometry the Sample A
(0.213 nm) is higher than Sample B (0.015 nm). From the preparation, Sample A is a raw
mushroom and Sample B is a cooked mushroom. Each mushroom loses up to half of its
nutrients when it is cooked. The protein will be denatured by heating.

Based on the graph, we can conclude the concentration of protein. The concentration
of protein in Sample A is 3.90 mg/ml and in Sample B is 0.98 mg/ml. So, it shows that the
concentration of Sample A is higher than Sample B. From the graph, the absorbance has a
direct relationship with the concentration of the protein sample, meaning the higher the
absorbance, the higher the concentration of the sample.

When during the experiments, there have some recommendation. First, ensure the
spectrophotometer down to zero point by the reagent blank since it can be a very big factor
for error in the experiment.

CONCLUSION

Throughout this experiment, I can conclude that the higher the absorbance, the higher
the concentration of the sample.
REFERENCE

Internet

 Hwang Sone. 1st April 2013. Protein Experiment. Retrieved from

http://biochemistrygirls.blogspot.my/2013/04/experiment-2-protein-experiment.html
 Maia Appleby. Does Cooking a Mushroom Deplete Its Vitamin Content? Retrieved from
http://healthyeating.sfgate.com/cooking-mushroom-deplete-its-vitamin-content-7548.html
 Michelle. 8th January 2011. Protein Assay by the Bradford Method. Retrieved from
https://www.scribd.com/doc/46499757/Protein-Assay-by-the-Bradford-Method