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

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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-Xanthoproteine.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

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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

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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

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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

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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

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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)

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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

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Chromatography Analysis

http://www.macalester.edu/~kuwata/Classes/200102/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