qualitative tests
TRANSCRIPT
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Proteins & Amino Acids in Saliva
Co. Cordero. Cruz. De Jesus
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Sakaguchi TestTest for the presence of guanidine
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Sakaguchi TestTest for the presence of guanidine
ARGININE
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Mechanism:
Sakaguchi TestReagents:
α-naphtholNaOH*NaOCl
+ NaOH, pH
zwitterionic form
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Sakaguchi Test
condensation reaction with
red/wine-colored solution
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For compounds containing a phenolic hydroxy groupAmino acid: Tyrosine
Compound must bevalidated as protein/amino acid to confirmpresence of tyrosine
Millon-Nasse Reaction
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Millon-Nasse ReactionMillon’s Reagent: Mercuric ion in acidMechanism:
Tyrosine + Millon’s Reagent = complex
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Millon-Nasse ReactionComplex treated with Nitrous Acid (NaNO2)
yields apink-red solution
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XANTHOPROTEIC REACTION
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XANTHOPROTEIC REACTION Boiling concentrated nitric acid reacts with
tyr, trp and phe to yield yellow products.This reaction involves the nitration of
benzene nucleus in alkaline medium. As a result, amino acids that contain aromatic nucleus undergo this reaction.
Aromatic AAs form yellow nitro derivative on heating with concentrated nitric acid, the salts of this derivative are orange.
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XANTHOPROTEIC REACTION
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(A) NITRATED TYROSINE AND TRYPTOPHAN (B)
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XANTHOPROTEIC REACTIONUsing 65% nitric acid the aromatic rings of
amino acids like tyrosine and tryptophan are nitrated. The nitro derivate show an intensely yellow color. Because nearly all proteins contain aromatics it is taken as a protein-test either.
The yellow stains on the skin caused by nitric acid are the result of the xanthoproteic reaction. The epidermis cells of the skin contain aromatic proteins.
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NINHYDRIN REACTION
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NINHYDRIN REACTIONTriketohydrindene hydrate,
commonly known as ninhydrin reacts with amino acids to form a purple colored imino derivative. This derivative forms a useful test for amino acids, most of which are colorless.
Ninhydrin is a powerful oxidizing agent which reacts with all amino acids between pH 4-8 to produce a purple-colored compund
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NINHYDRIN REACTIONA color reaction given by amino
acids and peptides on heating with the chemical ninhydrin
The amino acids proline and hydroxyroline also reacts but produces a yellow color.
Ninhydrin (triketohydrindene hydrate) is an oxidating agent which leads to the oxidative deamination of alpha-amino groups. It is very important for the detection and the quantitative analysis of amino acids.
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NINHYDRIN REACTIONNinhydrin also reacts with
primary amines however the formation of carbon dioxide is quite diagnostic for amino acids.
Alpha amino acids yield a purple substance that absorbs maximally at 570 nm. Imino acids (proline) yield a yellow product (absorption maximum 440 nm).
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NINHYDRIN REACTIONα-amino acid + 2ninhydrin CO2 + aldehyde + final complex(purple) +
3H2O Ninhydrin, which is originally yellow, reacts
with amino acid and turns deep purple color that is detected in this method.
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Biuret TestPresence of peptide bonds is detected by
performing a chemical test named biuret test.
The Biuret Reagent is made of sodium hydroxide and copper sulfate. The blue reagent turns violet in the presence of proteins, and changes to pink when combined with short-chain polypeptides.
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Cupric ion in an alkaline medium forms a violet coloured complex with peptide bond nitrogens of peptides and proteins.
The reaction is so named biuret(NH2CONHCONH2) formed by condensation of two molecules of urea, when heated at 180C, also answers this test. The minimum requirement for a positive test is the presence of 2 peptide bonds in the molecule.
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It yielded a positive result for saliva thus, saliva contains proteins.
It yielded a negative result for glycine.
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Saliva Glycine
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Bromine Water testThe bromine water test is an example of an
addition reaction.(A reaction in which a small molecule adds on across a double bond).
The decoloration of a solution of bromine in water is an analytical test for the presence of alkenes:
CH2=CH2 + Br2 → BrCH2-CH2Br
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Alkenes are able to undergo addition reactions because they contain a double bond.
They decolourise because they are unsaturated and have a carbon=carbon double bond
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Tryptophan
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Formation of pinkish layer is the positive result.
Tryptophan is positive under the bromine water test
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Pauly Reaction Diazotization
General Mechanism:
Sulfanilic acid gets diazotized in the presence of sodium nitrite (NaNO2) + sample + sodium carbonate (Na2CO3)
POSITIVE TEST : dark yellow or orange
: histidine and tyrosine residues
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Samples
Saliva : +
: contains histidine and tyrosine
Histidine : +
Tyrosine : +
* Theoretical result
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Saliva
HistidineTyrosine
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Lead Acetate Reaction
Liberates sulfur content to detect (cys)
Sulfur group of cysteine is liberated through heating with strong alkali
Treatment with alkali does not liberate sulfur from methionine
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POSITIVE TEST :gray : cysteine (cys)
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cys
saliva
hair
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* Theoretical result
Samples
Saliva : (-)
Hair : (-)
: high amount of sulfur due to cysteine
Cysteine: (+)
: sulfur was liberated
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Referencesn.a.(n.d.). Amino Acids. Date Retrieved August 2,2010,from http://home.earthlink.net/~dwyerg/HL%20Labs/proteins%20and%20amino%20acids.htm
n.a.(n.d.). Diazotization. Date Retrieved August 2,2010,from http://www.ecompound.com/Reaction%20reference/reactions/Diazotization%20related%20reactions.gif
n.a.(n.d.). Hair fibers. Date Retrieved August 3,2010,from http://www.keratin.com/aa/aa012.shtml