Protein Tests

PROTEINS AND

AMINO ACID ANALYSIS

Janir Ty DatukanDepartment of Physical Sciences

College of SciencePhilippine Normal University

OutlineColorimetric Test

Biuret TestNinhydrin TestXanthoproteic TestMillon-Nasse Test Hopkins-Cole ReactionSakaguchi ReactionLead Acetate Test

OutlinePrecipitation Reaction of Proteins

Heat and AcidAlcoholAlkaloidal ReagentsHeavy Metal SaltsIsoelectric PointSalting Out

Chromatography Analysis

Colorimetric Reactions

Biuret TestQuantitative photometrical determination

of total protein concentrationProtein must have at least three peptide

bondsPositive test produces a blue- to violet-

colored solutionIntensity of color depends on the number of

peptide bonds present in the sampleBiuret 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 Test2,2-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_reactions.png

Xanthoproteic TestThe 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 doesn’t undergo nitration because it’s 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 TestMillon-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 ReactionSpecific test for the indole ring in the

amino acid tryptophanHopkins-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


Sakaguchi ReactionA colorimetric reaction for identification

and quantification of guanidinium group of arginine

Reagent includes α–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 ReactionTests for the presence of sulfur from

cysteine and methionineReagent includes sodium hydroxide and

lead(II) acetateBoiling 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


Precipitation Reactions

Heat and AcidHeat disrupts hydrogen bonds of

secondary and tertiary protein structureThe primary structure remains unaffectedThe protein increases in size due to

denaturation and coagulation occursAddition of acetic acid to albumin results

in the formation of a cloudy white substance called a coagulum

AlcoholHydrogen bonding occurs between amide

groups in the secondary protein structureHydrogen 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 ReagentsAlkaloidal 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 SaltsHeavy metals (e.g. Hg2+, Pb2+, Cu2+) are high

molecular weight cationsThe positive charge of these cations

counteracts the negative charge of the carboxylate group in proteins

This results in the formation of a precipitateHeavy 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


Isoelectric PointThe solubility of protein depends on the pH of

the solutionIt is positively charged at low pH and

negatively charged at high pHThe 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 moleculesTherefore, a protein is the least soluble when

the pH of the solution is at its isoelectric point

Salting OutProtein molecules contain both

hydrophilic and hydrophobic amino acidsIn 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 OutIn 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.macalester.edu/~kuwata/Classes/2001-02/Chem%2011/Revised%20Amino%20Acids%20(9%201%2001).pdf


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

Protein Tests

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