Janir Ty Datukan

Department of Physical Sciences

College of Science
Philippine Normal University
Outline
Colorimetric Test
Biuret Test
Ninhydrin Test
Xanthoproteic Test
Millon-Nasse Test
Hopkins-Cole Reaction
Sakaguchi Reaction
Lead Acetate Test
Outline
Precipitation Reaction of Proteins
Heat and Acid
Alcohol
Alkaloidal Reagents
Heavy Metal Salts
Isoelectric Point
Salting Out
Chromatography Analysis
Biuret Test
Quantitative photometrical determination of total
protein concentration
Protein must have at least three peptide bonds
Positive test produces a blue- to violet-colored
solution
Intensity of color depends on the number of
peptide bonds present in the sample
Biuret Reagent is made from potassium hydroxide
and hydrated copper(II) sulfate
 Biuret Test

http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/D-Biuret-e.htm
Ninhydrin Test
2,2-Dihydroxyindane-1,3-dione is a chemical used
to detect ammonia or primary & secondary amines
Positive test results in deep blue or purple color
known as Ruhemann's purple
Commonly used to detect fingerprints due to the
terminal amines or lysine residues in peptides and
proteins
 Ninhydrin Test

http://homepages.ius.edu/dspurloc/c122/casein.htm
http://3.bp.blogspot.com/_as7Ap63dYXM/TEme08c92EI/AAAAAAAABXQ/0cSba6RhaQY/s1600/ninhydrin_reaction
s.png
Xanthoproteic Test
The aromatic groups in the amino acids can
undergo nitration with nitric acid and give in
yellow-colored products
Only phenyl rings containing an activating group
can be nitrated
Phenylalanine doesnt undergo nitration because
its not activated like tryptophan and tyrosine
 Xanthoproteic Test

http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/D-Xanthoprotein-
e.htm

http://nuwanthikakumarasinghe.blogspot.com/2011/05/tests-for-proteins-2.html
Millon-Nasse Test
Millon-Nasse reagent is made from by dissolving
metallic mercury in nitric acid and diluting with
water
May also be prepared from mercury metal
dissolved in H2SO4
Detects phenolic groups, which means only for
tyrosine
Positive result produces a red-brown solution or
precipitate
 Millon-Nasse Test

http://www.drugs.com/dict/millon-nasse-test.html
Hopkins-Cole Reaction
Specific test for the indole ring in the amino acid
tryptophan
Hopkins-Cole reagent contains glyoxylic acid (Mg
powder, oxalic acid, acetic acid) and concentrated
H2SO4
Positive reaction will show a purple-colored ring in
the solution boundaries
 Hopkins-Cole Reaction

http://nuwanthikakumarasinghe.blogspot.com/2011/05/tests-for-proteins-2.html
Sakaguchi Reaction
A colorimetric reaction for identification and
quantification of guanidinium group of arginine
Reagent includes naphthol and sodium
hypobromite (NaBrO) or bromine water in alkaline
solution
Positive test results in an orange- to red-colored
solution
 Sakaguchi Reaction

https://upload.wikimedia.org/wikipedia/commons/f/f8/Sakaguchi_reaction.svg
Lead Acetate Reaction
Tests for the presence of sulfur from cysteine and
methionine
Reagent includes sodium hydroxide and lead(II)
acetate
Boiling with NaOH converts S in the amino acid to
NaS, which then precipitates as balck PbS with the
addition of lead acetate
Positive result is a black solution or precipitate
 Lead Acetate Reaction

http://nuwanthikakumarasinghe.blogspot.com/2011/05/tests-for-proteins-2.html
Heat and Acid
Heat disrupts hydrogen bonds of secondary and
tertiary protein structure
The primary structure remains unaffected
The protein increases in size due to denaturation
and coagulation occurs
Addition of acetic acid to albumin results in the
formation of a cloudy white substance called a
coagulum
Alcohol
Hydrogen bonding occurs between amide groups
in the secondary protein structure
Hydrogen bonding between "side chains" occurs in
tertiary protein structure in a variety of amino acid
combinations
All of these are disrupted by the addition of
another alcohol
 Alcohol

http://www.elmhurst.edu/~chm/vchembook/568denaturation.html
Alkaloidal Reagents
Alkaloidal reagents (e.g. tannate &
trichloroacetate) are high molecular weight anions
The negative charge of these anions counteracts
the positive charge of the amino group in proteins
This results in the formation of a precipitate
Heavy Metal Salts
Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high
molecular weight cations
The positive charge of these cations counteracts
the negative charge of the carboxylate group in
proteins
This results in the formation of a precipitate
Heavy metals may also disrupt disulfide bonds
because of their high affinity and attraction for
sulfur
This will also lead to the denaturation of proteins
 Heavy Metal Salts

http://www.elmhurst.edu/~chm/vchembook/568denaturation.html
Isoelectric Point
The solubility of protein depends on the pH of the
solution
It is positively charged at low pH and negatively
charged at high pH
The pH at which a protein molecule has a net
charge of zero is called the isoelectric point
In general, the net charge, either positive or
negative, can interact with water molecules
Therefore, a protein is the least soluble when the
pH of the solution is at its isoelectric point
 Salting Out
Protein molecules contain both hydrophilic and
hydrophobic amino acids
In aqueous medium, hydrophobic amino acids
form protected areas whil hydrophilic amino acids
form hydrogen bonds with surrounding water
molecules (solvation layer)

http://www.pua.edu.eg/Version2/Courses2/Dentistry%20Courses/Freshmen/Spring/BCM101/Practical/Week%204%
20practical%20_Chemistry%20of%20proteins_.pdf
Salting Out
In salt solutions (e.g. ammonium sulfate), some of
the water molecules in the solvation layer are
attracted by salt ions
When salt concentration gradually increases, the
number of water molecules in the solvation layer
gradually decreases
The protein molecules then coagulate forming a
precipitate; this is known as salting out
 Protein Precipitation

http://www.elmhurst.edu/~chm/vchembook/568denaturation.html
 Chromatography Analysis

http://www.macalester.edu/~kuwata/Classes/2001-
02/Chem%2011/Revised%20Amino%20Acids%20(9%201%2001).pdf
 Chromatography Analysis

http://faculty.buffalostate.edu/wadswogj/courses/BIO211%20Page/lectures/lab%20pdf's/Amino%20Acid%20lab.pdf