KSU - College of Science - Department of Biochemistry BCH 233, Zaenab Alzahrani, 2nd semester, 2009-2010

Physical Biochemistry Lab

Experiment no. 6:

Biuret assay
Objective:
To estimate the protein concentration in a given sample.

Introduction and principle:

The biuret reaction can be used for both qualitative and quantitative analysis of protein. The
biuret method depends on the presence of peptides bonds in proteins. When a solution of
proteins is treated with cupric ions (Cu2+) in a moderately alkaline medium, a purple colored
Cu2+ - peptide complex is formed which can be measured quantitatively by spectrophotometer
in the visible region. So, biuret reagent is alkaline copper sulfate solution.

Cu2+ - peptide complex

The intensity of the color produced is proportional to the number of peptide bonds that are
reacting, and therefore to the number of protein molecules present in the reaction system.
The reaction don't occur with amino acids because the absence of peptide bonds, and also
that with di-peptide because presence of only one peptide bond, but do with tri-, oligo-, and
poly-peptides. Biuret reaction needs presence of at least two peptide bonds in a molecule.
The reaction occurs with any compound containing at least two bonds of:

-HN-CO- , -HN-CH2- , -HN-CS-

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KSU - College of Science - Department of Biochemistry BCH 233, Zaenab Alzahrani, 2nd semester, 2009-2010

The reaction takes its name "Biuret Reaction" from the fact that biuret itself, obtained by
heating urea, gives a similar colored complex with cupric ions.

In this experiment the amount of isolated myoglobin protein from the skeletal muscle in last
experiment is determined by biuret assay and from the standard curve of bovine serum
albumin (BSA). A standard curve is a quantitative research tool; it is a method of plotting
assay data that is used to determine the concentration of a substance like protein. First you
perform a biuret assay with various known concentrations of bovine serum albumin. The
response is absorbance of the colored product. Graph these data to make a standard curve,
concentration on the X axis, and the absorbance on the Y axis. Also perform the biuret assay
with your sample. You want to know the concentration of the myoglobin in sample. To analyze
the data, fit a line or curve through the standards. For the sample, read across the graph from
the spot on the Y-axis that corresponds to absorbance of the sample until you intersect the
standard curve. Read down the graph until you intersect the X-axis. The concentration of
myoglobin in the sample is the value on the X-axis.

Material and apparatus:

1. Protein sample of unknown concentration (from previous experiment)

2. Standard BSA (5 g/l)
3. Distilled water
4. Biuret reagent
5. Test tubes
6. Label
7. Test tube rack
8. Pipettes
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KSU - College of Science - Department of Biochemistry BCH 233, Zaenab Alzahrani, 2nd semester, 2009-2010

9. Pipette bulb
10. Vortex mixer
11. Spectrophotometer
12. Cuvettes

Methods:

1) Label 9 test tubes as (1 to 9) and place them in a test tube rack.

2) Add to each tube the solutions in the following table:

Test tubes
Amount of each solution Sample in
Standards blank
by (ml) duplicate
1 2 3 4 5 6 7 8 9
Standard BSA 0.1 0.2 0.4 0.6 0.8 1 - - -
Distilled water 0.9 0.8 0.6 0.4 0.2 - - - 1
Sample - - - - - - 1 1 -
Biuret reagent 4 4 4 4 4 4 4 4 4

3) Mix well by vortex mixer and allow standing for 20 min.

4) Read the absorbance for each tube against the blank at 540 nm.
5) Calculate the protein concentration in each tube of standard.
6) Record your result in the following table:

Test BSA conc. Absorbance

tube (g/l) at 540 nm
1
2
3
4
5
6
7
8

7) Plot the standard curve using concentration of standard tubes of BSA (g/l) against the
absorbance at 540 nm.

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KSU - College of Science - Department of Biochemistry BCH 233, Zaenab Alzahrani, 2nd semester, 2009-2010

8) Calculate the mean of absorbance of the duplicate sample and obtain the concentration
of myoglobin in the sample from the standard curve.
9) Find out the protein concentration (g/g) in the original skeletal muscle tissue sample.
(review the previous experiment "salting in, salting out, & dialysis")

Questions:

1- Explain why you measure the absorbance at 540 nm in this experiment? And what type
of cuvette did you use?
2- Mention the alternative method to estimate the protein concentration in a given sample
in case of cannot apply standard curve in biuret assay?

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