Investigating the concentration of protein in different conditions

Introduction- Proteins are essential form of nutrients and they help in growth,
repair and can form enzymes. They are made from amino acids linked
together by covalent peptide bonds. Peptide bonds are formed when
dehydration reaction occurs between two amino acids eliminating the water
molecule to a large molecule. So in order to find the concentration of protein
from the absorbance reading, an experiment was carried out in two parts for
different conditions. The number of peptide bond in a solution was measured
with the biuret reagent from the intensity of the colour of the complex
formed. So the number of protein in the sample can be found.
Part1. A standard curve was constructed for the unknown sample provided,
as the standard curve was used in this experiment to calculate the
concentration of an unknown solution, by using the protein concentration
that corresponds to the absorbance reading for the unknown solution. For
plotting the curve six test tubes were used where different amount of buffer
and BSA protein solution was added, with the biuret reagent. Then total
volume and final protein concentration was calculated along with the
absorbance and the unknown concentration from the standard curve that
was constructed.
Part2.Two different suspension of silver beet leaf were used to measure the
different concentration of protein and the absorbance.
The null hypothesis is, the plant raised in sun could have more rubisco than
the one raised in the shade
Method- Part1- 8 test tubes were taken and labeled for this experiment from
1-8. Then buffer was pipetted into test tube 1-6, and the amount added was
mentioned in the table. The pipette used was changed and a new pipette
was used to added volumes of protein solution as per the table into test
tubes 1-6.Then 4ml of biuret reagent was added for each test tube from an
auto dispenser, in a fume cupboard. The final volume of each test tube at
this point was 5ml the test tubes were all capped and the contents were
mixed by inverting them and, it was left at room temperature for 25mins .
Once the colour was stable the absorbance reading was carried out.
Part2- The test tubes labeled as 7 and 8 were used for this experiment , one
was labeled as Sun and the other as Sh for shade. In tube 7 Sun
suspension was pipetted and in the test tube 8 Shade suspension. Then
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4ml of biuret solution was added to each rest tube and the cap was corked
and the suspension inverted and mixed properly and left for 25 minutes for
the colour to develop and stabilize. Then the absorbance reading was
carried out, but as the absorbance reading was to high to fit in the graph, the
sample of protein solution was take was i/4th ml of the original 1ml and the
diluted by 3/4th of the buffer so that the total volume was 1ml. Then the
biuret reagent was added capped and inverted to miss the solution and the
left for 25 minutes for the colour to stabilize. Then the absorbance reading
was measured using the spectrometer.

Absorbance- For the absorbance reading spectrometer was set up and used.
The solutions in the test tubes 1-8 are poured in the cuvette which was
washed and dried first. After pouring the solutions the cuvette is wiped clean
of any hand prints and the wider sides are facing the light. The first reading
was of a reagent blank, which gave the protein concentration reading as
zero, so that the amount of colour produce was automatically subtracted
from the test tubes 2-8.
In this way all the readings of the absorbance was take and recorded.

Results

Tubes
1

Buffer(ml)
BSA Protine
solution(10mg ml-1)
(ml)
Biuret reagent (ml)
Total volume (ml)
Final Protein
concentration(mg ml-1)
Absorbance

1.0
0.0

2
0.8
0.2

3
0.6
0.4

4
0.4
0.6

5
0.2
0.8

6
0.0
1.0

4.0
5.0
0.0

4.0
5.0
2.0

4.0
5.0
4.0

4.0
5.0
6.0

4.0
5.0
8.0

4.0
5.0
10.0

0.45
4

0.570

0.00 0.11
0
4

0.19 0.33
8
4

Fig: Table1-Experimental protocol for construction of the protein standard

curve

Sun

Protein solution (ml)

Biuret reagent(ml)
Absorbance
Protein concentration derived from
Standard curve(mg ml-1)
Protein concentration of original
unmodified solution (mg ml-1)

Shad
e

Procedure
1
1
4
4

Columns for modified

procedure
0.25
0.25
4
0.551
9.8
3.92

Fig: Table 2- Experimental protocol for determining the protein

concentration of silverbeef in sun and shade

4
0.359
6.15
1.48

Protein concentration vs absorbance

0.6
0.5
0.4
absorbance

0.3
0.2
0.1
0
0

10

12

protein concentration (mgml-1)

From the results it was clear that the aim and hypothesis was proved right,
the absorption reading increased when protein concentration increased.
When the protein concentration was 2, 8 and 10 the absorption reading was
0.114, 0.454 and 0.570 respectively. Even that Sun and Shade showed the
results clearly. The protein concentration was 9.80 and 6.15 and absorption
was 1.36 and 1,21 respectively.
Discussion- From the graph, tables and results it was interpreted that as the
protein concentration increased the absorbance rate increased, when the
protein concentration in table 1 was 2ml , 4ml and 10 ml the absorbance
was 0.114, 0.198 and 0.570 respectively. Even from the graph and table 2
the sun and shade values its clear that the plants in sun had more
protein and they had higher absorbance reading.
The graph that was drawn was a best fit line so that the anomalies were
avoided. There would be experimental errors, for example if the measured
absorbance reading is off the scale of y-axis there will be no possible way to
determine the concentration of protein in the solution from the standard
curve even if it is increasing gradually, but it could be predicted by
extrapolation of the curve, or the reading would be affected due to the
wiping cuvette, when the cuvette still has hand prints or water in it, or due to
parallax error.
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For this experiment the absorbance reading for the Sun is higher than the
Shade, 1.367 and 1.210 respectively, since they cannot be determined from
the standard curve the experiment must be modified so that the absorbance
value lies within the range of y-axis. So in order to modify it, the
concentration of Sun and Shade was reduced to 0.25ml and diluted with
0.75ml of buffer solution. The 4ml biuret reagent that was added in the
original experiment was kept same for this modified one.
From the results in table1 it was clear that as the concentration of protein
increase the absorption value increased, so increasing the concentration of
BSA protein in tube 1-6 ensured that the absorbance reading also increased.
This would increase the absorbance range of the standard curve.
But due to a lot of biological reasons like there would be different form of
proteins so the reading would vary, or the silverbeet leaves had different
shaped so the concentration was different so the results varied.

Conclusion- The hypothesis was proven correct but by improving the method
an accurate result could have been obtained
Reference
Reeca, Cain, J.B, M.L, 2014. Campbell Biology. 5th ed. Australia: pearson.
Layne, E. 1957. Spectrophotometric and turbidimetric methods for
measuring proteins.
Hartree, E.E. 1972. Determination of protein: A modification of the Lowry
method that gives a linear photometric response. Anal. Biochem. 48:422427.Methods Enzymol. 3:447-455