SBL 1023

Technique in Biology And

Biochemistry Laboratory

Lab 3: Spectrophotometry And

Protein Concentration Measurement

NOR AIYNA SYERA BT MOHD ASRI

STUDENT`S NAME
( E20161015981)

GROUP B

Lab 3: Spectrophotometry and protein

EXPERIMENT`S NO
concentration measurement

DATE/DAY/PLACE 21/11/2017 /TUESDAY/ B2-L3-MP 14

LECTURE`S NAME Professor Madya Dr. Shakinaz Binti Desa

Title

- Experiment 3: Spectrophotometry (protein analysis in different type of mushroom).

Objective

- To use the Spectrophotometer correctly.

- To analysis the amount of protein in a sample.
- To compare the amount of protein in two different condition of mushroom with the
same type.

Introduction

Proteins are polymers of amino acids. Twenty different types of amino acids occur
naturally in proteins. Proteins differ from each other according to the type, number and
sequence of amino acids that make up the polypeptide backbone. As a result they have
different molecular structures, nutritional attributes and physiochemical properties. Proteins
are important constituents of foods for a number of different reasons. They are a major source
of energy, as well as containing essential amino-acids, such as lysine, tryptophan,
methionine, leucine, isoleucine and valine, which are essential to human health, but which the
body cannot synthesize. Proteins are also the major structural components of many natural
foods, often determining their overall texture, e.g., tenderness of meat or fish products.
Isolated proteins are often used in foods as ingredients because of their unique functional
properties, i.e., their ability to provide desirable appearance, texture or stability. Typically,
proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food
proteins are enzymes which are capable of enhancing the rate of certain biochemical
reactions. These reactions can have either a favourable or detrimental effect on the overall
properties of foods. Food analysts are interested in knowing the total concentration, type,
molecular structure and functional properties of the proteins in foods.

Biuret protein assay use biuret reagent that detect 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.
 Figure 1. Biuret reagent is a blue liquid that changes to purple when proteins are present

They are various ways to determine the concentration of protein. But it depends on
the specificity, sensitivity, the measurable range of concentration, the accuracy, the nature of
the protein to be examined, the presence of materials interfering with the measurement, and
the time required for the measurement.

Equipment and materials

- Stock solution of Bovive Serum Albumin (BSA): 10mg/ml

- Deionized water (dH2O)
- Test tubes and stand
- Pipette -Biuret reagent
- Spectrophotometer
- Protein samples

Procedure

A) Protein preparation
- 1 sets of test tubes with the numbers 1 to 6 and the gelatine stock solution (10 mg/ml)
was prepared according to the concentration listed below:
 gelatine
BSA stock
Tube conc. H2O (ml)
(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 1.0: preparation of stock solution

- Duplicate test tubes was prepared for protein samples and 1ml of the protein samples
was carefully pipetted into each tubes.
- 2ml of biuret reagent was added to every tube, the 7 tubes for the standard curve and
the duplicate tubes for protein samples.
- The tubes was covered with parafilm and briefly vortex to ensure that the protein
samples and the biuret reagent are thoroughly mixed.
- Allowed the tubes to stand at 15 minutes.
- The spectrophotometer was switch on and adjusted the wavelength to 550nm.

B) Determined protein concentration

1. 1ml of solution from tube 1 was transferred into a cuvette and gently wipe the
cuvette with a paper towel to remove fingerprints and dust.
2. The absorbance was set to 'zero'. This tube will serve as 'blank'.
3. The absorbance of the other standards and sample proteins was measured used
the step as in (1).
4. The absorbance of each standards and sample was recorded.
5. A graph of standard curve was plotted using the absorbance value of protein
standards and interpolate with the concentration of protein sample.
Result

Raw
mushroom

Boiled
mushroom

Test tube BSA concentration Absorbance

(mg/ml)

1 0 0.000
2 1 0.016
3 2 0.066
4 3 0.072
5 4 0.221
6 5 0.278
7 6 0.500
8 Raw 0.213
9 Boiled 0.015
Table 1: Result for the experiment
 Table 2: observation on the test tube 1-6

Graph

Absorbance of Protein Sample

0.6

0.5

0.4 raw
absorbance

boiled
0.3
Series1
0.2

0.1

0
0 1 2 3 4 5 6 7 8 9 10
concentration
Discussion

In this lab, we used two method to determine the concentration of protein which are
Biuret test and spectrometry test. Biuret test was used to detect the presence of peptide bond.
It was treated with copper sulphate solution. Biuret test in presence of alkali, protein reacts
with copper (II) ions, (blue in colour) to form a violet coloured complex called biuret. While,
in this lab we used mushroom as a sample, so after we added 1 ml of sample solution into the
test tube contained the biuret reagent, gelatine and distilled water, it show negative result, no
violet colour was present. Instead, the colour was change to dark blue greenish in colour, due
to green pigment on the mushroom. Besides, biuret test was low sensitivity and that it
requires at least 1 mg of protein to be tested.

From the result of the experiment, we can see that the raw mushroom has more protein
than boiled mushroom. This in turns cause the graph with raw mushroom as a sample has a
higher absorbance than the one with the boiled mushroom as the sample. This is because
when the mushroom was boiled in 100oC, the protein is lost and decomposed when heated.

Conclusion

For the conclusion, the raw mushroom has more protein than the boiled mushroom. For the
vegetarian people, they can eat the mushroom without boiled it to get the more protein from
the mushroom. They also can boiled it but just for a few seconds to prevent the protein from
lost and decomposed.

References

1. Marietta, 2017, Measuring Protein Concentration Through Absorption

Spectrophotometry,
http://w3.marietta.edu/~spilatrs/biol309/labexercises/Spectrophotometry.pdf.
Retrieved on 3/12/2017
2. Wikipedia, 3 December 2017, Gelatine, https://en.wikipedia.org/wiki/Gelatin .
Retrieved on 3/12/2017
3. Lab 3: Spectrophotometry. (2017). SBL 1023 LAB MANUAL. Faculty science and
mathematic.