Protein

Biuret test for protein

Biuret test is a chemical procedure which is used to detect the present
of peptide bonds between amino acids in the protein molecules by using
biuret reagent. This reagent is composed of sodium hydroxide, copper
sulphate pentahydrate and sodium potassium tartrate. The present of
tartrate in the reagent will stabilize the cupric ion (Cu2+) and makes the
solution relatively stable compared with the one without it. Otherwise, it has
to be freshly prepared to be used. Cu2+ from copper sulphate pentahydrate
gives off the blue colour to this reagent.This biuret reagent usually is stable
for only few months but its colour may be darkened and some precipitate will
form. In order to keep it stable indefinitely, one gram of potassium iodide per
liter must be added and it has to be kept in a dark place.
If the peptide bonds are present, Cu2+ will form a violet complex
solution. As Cu2+ will only effectively react with peptide bonds in alkaline
medium, sodium hydroxide plays the main role to provide it. The peptide
bond of protein is oxidized first which the part where the amino group will
release a hydrogen atom and reduce a hydroxide ion of alkaline solution.
Cupric ion (Cu2+ ) is then reduced to cuprous ion (Cu+) and an electron is
released from the peptide bonds. A purple complex with the maximum
wavelength, max of 540 nm will be formed. According to Beers law,
absorbance of a compound is equal to its concentration, so biuret reaction is
suitable for assay the concentration of protein by spectrometric method. This

is because peptide bonds are occuring at the same frequency per amino acid
in the peptide chain.

NH2

C
H

C
O

R
H

H NH2
O

HC

C
H

ON

NR

O
C

OH

In the alkaline solution,

NH2

H
C

C OH-C

OH H2O

R
OHH

NH2

NH2

In the present of cupric

ions
H

Cu2+
OH

Cu

OH

Methods
1.
2.
3.
4.

A set of 6 tubes was prepared and labeled with the wax pencil.
2 ml of each sample was added into each test tube.
Then, each of the tube was added by 2 ml of biuret reagent.
The content of each tube was mixed thoroughly by using the vortex

genie.
5. The mixtures are left aside about 2minutes to react.
6. Each of the tube was examined carefully. All the colour changes were
recorded.
7. The observations were tabulated in Table 2 by indicating the presence
(+) and absence (-) of peptide bonds in each sample.

Ref: http://www.piercenet.com/method/chemistry-proteinassays#copperassays
http://fulltimes.wordpress.com/protein/