Biuret Test

1. B. 2 COLOR REACTIONS OF PROTEINS AND AMINO ACIDS
- BIURET TEST
Castro, Elvin Clyde
Dilangalen, Alezandra
Malaluan, Harold Jhon
Panes, Andrew
Pimentel, Anne Lexcee
Ragasa, Alyssa
Villanueva, Love
BSRT- 1C Group 4
Introduction

2. Proteins are essential nutrients for the human body. They are one of the building blocks
of body tissue and can also serve as a fuel source. Proteins are large biomolecules, or
macromolecules, consisting of one or more long chains of amino acid residues. Proteins
perform a vast array of functions within organisms, including catalysing metabolic
reactions, DNA replication, responding to stimuli, providing structure to cells and
organisms, and transporting molecules from one location to another. Proteins differ
from one another primarily in their sequence of amino acids, which is dictated by the
nucleotide sequence of their genes, and which usually results in protein folding into a
specific three-dimensional structure that determines its activity.
https://ghr.nlm.nih.gov/primer/howgeneswork/protein
The biuret test is a chemical assay that detects the presence of proteins in a sample.
The test relies on a color change to confirm the presence of proteins. If proteins are
found, the sample will turn violet. The biuret test uses an alkaline mixture, or reagent,
composed of potassium hydroxide and copper sulfate. The normal color of biuret
reagent is blue. The reagent turns violet in the presence of peptide bonds -- the
chemical bonds that hold amino acids together. The proteins detected must have at
least three amino acids, which means that the protein must have at least two peptide
bonds. The reagent’s copper ions, with a charge of +2, are reduced to a charge of +1 in
the presence of peptide bonds, causing the color change. The techniques of absorption
spectroscopy, which identify the electromagnetic frequencies a sample will absorb,
allow testers to quantify the concentration of protein in a sample.
For every procedure
2.1 Egg white and aspartame

3. i. Objective: To detect the presence of two or more peptide bonds in a solution
ii. Presentation of results
The visible result when biuret reagents mixed with egg white, displayed a violet ring
coagulation. On the other hand, when biuret reagents were mixed or added with
aspartame solution, it gave a color blue to the solution.
2.2 Total Protein and Albumin
i. Objective : To detecting the presence of peptide bonds
ii. Presentation of Result:
Total Protein(TP)
The solution when mixed with copper sulfate turns from blue to violet (deep purple),
indicating the presence of protein in the solution.
Albumin (A)
The solution turns blue to pink which determines that peptide are present in the solution.
iii.
A. What principle involved in the Biuret Test?

4. When biuret is treated with dilute copper sulfate in alkaline medium, a purple colored
compound is formed. It is believed that the color is due to the formation of a copper co-
ordinated complex or chelate complex. Cupric ions or Cu (II) ions form a violet-colored
chelate complex with unshared electron pairs of peptide nitrogen and oxygen of water.
The chelate complex absorbs light at 540 nm and hence appears violet. The color
change from blue to violet indicates the presence of proteins.
The greater the number of peptide bonds in a protein, the greater the color intensity.
Hence, the color change is from blue to pink if the concentration of peptide bonds is low
like in short-chain peptides. The principle of biuret test is conveniently used to detect
the presence of proteins in biological fluids
B.What is the Role of Reagents in the test?
The biuret test uses an alkaline mixture, or reagent, composed of potassium hydroxide
and copper sulfate. The normal color of biuret reagent is blue. The reagent turns violet
in the presence of peptide bonds -- the chemical bonds that hold amino acids together.
The reagent’s copper ions, with a charge of +2, are reduced to a charge of +1 in the
presence of peptide bonds, causing the color change
C.Will all proteins give a positive biuret test? why?
All proteins will give a positive result to Biuret Test because all proteins have peptide
bonds. However, not all peptides will give a positive result because in order to have
positive results, a compound must have two or more peptide bond and since not all
peptide has a two or more bond.
D. Account the difference between eggwhite and aspartame.
Account for compound containing two or more peptide bonds give a characteristic
purple color. Egg white is positive for the test because the structure of the egg white

5. shows many peptide bonds while the aspartame gave the color blue since aspartame is
a dipeptide, it will be negative to biuret test in which it shows that aspartame has only
one peptide bond.
E. What is the function of 23% Na2SO4?
Protein stability is the net balance of forces, which determine whether a protein will be
its native folded conformation or a denatured (unfolded or extended) state. The net
stability of proteins is quite small and is the difference between two large opposing
forces. The folded native state of protein structures is stabilized by various atomic/group
interactions, such as hydrophobic, electrostatic, hydrogen bonding, van der Waals, and
disulphide, and the unfolded state is dominated by entropic and nonentropic free
energie, and that’s how sodium sulfate stabilize the protein.
F. What is the function of ether?
Ether denatures proteins by disrupting the side chain intramolecular hydrogen bonding.
New hydrogen bonds are formed instead between the new alcohol molecule and the
protein side chains
G. the TP (total protein) showed violet (deep purple) color indicating the presence of
proteins and in the A (albumin) showed blue reagent changed to pink when combined
with short-chain polypeptides which contain smaller numbers of peptide bonds.
H. Account difference in color intensity between samples TP and A.
Biuret reagent, made of sodium hydroxide and copper (II) sulfate, is used for
determining the presence of protein in a sample. The test relies on the reaction between
copper ions and peptide bonds in an alkaline solution. A violet color indicates the

6. presence of proteins. Proteins give a strong Biuret reaction because they contain a
large number of peptide bonds. The blue reagent changes to pink when combined with
short-chain polypeptides which contain smaller numbers of peptide bonds.
I. Give the importance of biuret test in protein hydrolysis.
When a protein is hydrolyzed, some or all of the peptide bonds are broken. The
products of partial hydrolysis are peptides and the products of complete hydrolysis are
amino acids. Biuret Test. Compounds that contain two or more peptide bonds will react
with Cu2+ in a basic solution to form a violet-pink complex.In the presence of peptides,
a copper(II) ion forms mauve-colored coordination complexes in an alkaline solution.
The test is named so because it also gives a positive reaction to the peptide-like bonds
in the biuret molecule. In this assay, the copper(II) binds with nitrogens present in the
peptides of proteins.
References
K.S Publishing, (2009). Protein and Their function. Retrieved from
https://ghr.nlm.nih.gov/primer/howgeneswork/protein
Education Shuttle. Retrieved from https://education.seattlepi.com/biuret-test-mean-
biology-4659.html

7. Biuret Test. Retrived from http://onlinesciencenotes.com/biuret-test-principle-
requirements-procedure-and-result-interpretation/
Hillsborough Community College, (2001). Indicator Tests for Important Nutrients.
Retrived from https://www.hccfl.edu/media/571427/3-
indicator%20tests%20for%20important%20nutrients.pdf
ScienceDirect, (2002). Medicine and Dentistry. Retrieved from
https://www.sciencedirect.com/topics/medicine-and-dentistry/biuret