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Lab Report Experiment 3 (Spectrophotometry)

Determining Protein Concentration of Unknown Solutions :

The Biuret Protein Assay

Introduction

Why should you care about protein concentrations in solutions? There are a number of
reasons. In many lab experiments, for example, determining the amount of protein
biomolecules in a given solution is essential. The objectives of this experiment are to
determine the protein concentrations of an unknown solution using the Biuret method of
protein assay, and to learn how to use spectrophotometer in measuring the absorbance of
different protein concentrations. The Biuret method requires the solution being tested be
mixed with a Biuret reagent and run through a spectrophotometer.

Methods

For this experiment, five test tubes, each filled with different concentrations of bovine
serum albumine (BSA) ; 0 mg/ml, 2 mg/ml, 5 mg/ml, 8 mg/ml and 10 mg/ml, and labeled
as tube 1, 2, 3, 4, and 5 respectively. The first tube containing only distilled water acts as
reference or control. The dilution was done by bringing the total volume to 1.00 ml with
distilled water. 4 ml of Biuret reagent was then added to each tube and being vortex
immediately after that. The solutions were left for about 30 minutes. To determine the
absorbance of these solutions, a small amount of each solution was placed in a
spectrometer and the absorbance readings were taken, ranging from 0 to 0.7. Using the
concentration data and absorbance data of BSA protein, a standard curve can be
constructed, which is presented as the graph of absorbance vs concentration.
In the second part of this experiment, the unknown samples were prepared with different
concentrations using the techniques of 1, 1/2, 1/10 and 1/100. Each solution was then
being added with Biuret reagent and immediately vortex, then left for about 30 minutes.
Next, the level of absorption was recorded using a spectrophotometer. Finally,
concentrations of the unknowns were estimated using the standard curve plotted using
data from the first part of the experiment.
Results

Absorbance Corrected
Concentration of at 550nm Absorbance
Tube protein (mg/ml) (AU) (AU)
1 (reference) 0 0.1787 0.0000
2 2 0.2868 0.1081
3 5 0.5787 0.4000
4 8 0.6816 0.5029
5 10 0.8383 0.6596
Unknown 1 1.4625 1.2838
Unknown 1/2 1.4438 1.2651
Unknown 1/10 0.736 0.5573
Unknown 1/100 0.2819 0.1032

Absorbance vs concentration
0.8000

0.6000
Absorbance (AU)

0.4000

0.2000

0.0000
0 2 4 6 8 10 12
Concentration (mg/ml)

Figure 1 : Absorbance vs concentration graph.

Concentration of unknown 1/10 = mg/ml

Concentration of unknown 1/100 = mg/ml
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Discussion

Proteins are polymers made up of amino acids joined by peptide bonds. These peptide
bonds have partial double bonds character and absorb light. These feature is also
important in measurement of absorbency. Biuret reagent contains Cu2+ ion which form
complex with peptide bonds of the protein. The Biuret method is not highly sensitive but
it is the most linear because its colour depends on a direct complex between Cu 2+ and
peptide bonds of the proteins. Thus, the colour may slightly different depending on the
concentrations of the proteins.
A standard curve (Figure 1) was graphed using data from the five samples of known
absorbance and concentration. Unknown solution 1/10 and unknown solution 1/100 have
absorbance of 0.5573 and 0.1032 respectively, when ran through the spectrometer. These
points are then plotted on the y-axis of the graph and a line is drawn to the curve. The
intersection of both points show the protein concentration of unknown 1/10 as mg/ml
and unknown 1/100 as mg/ml. However, concentrations of unknown 1 and unknown
1/2 could not be determined using this standard curve because the absorbance for both
unknowns are more than the range of recorded absorbance of five samples of BSA.

Conclusion

From the standard curve plotted using the data recorded in this experiment, it can be
concluded that the amount of absorbance is directly proportional to the concentration of
proteins. In other words, the higher the concentration of protein, the higher the level of
absorption. Also, it is dependant on the amount of water in the solution. When the
amount of water increases, the concentration will decrease, thus the absorbance will also
decrease. By using the standard curve from data of this experiment, it is easier to
determine the concentration of unknown protein solution by first determining its level of
absorption.

Reference

1. http://www.iscid.org/encyclopedia/Protein_Assay
2. http://www.freeonlineresearchpapers.com/node/257
3. Lab Manual