Proteins & Amino Acids in Saliva

Co. Cordero. Cruz. De Jesus

Sakaguchi TestTest for the presence of guanidine

Sakaguchi TestTest for the presence of guanidine

ARGININE

Mechanism:

Sakaguchi TestReagents:

α-naphtholNaOH*NaOCl

+ NaOH, pH

zwitterionic form

Sakaguchi Test

condensation reaction with

red/wine-colored solution

For compounds containing a phenolic hydroxy groupAmino acid: Tyrosine

Compound must bevalidated as protein/amino acid to confirmpresence of tyrosine

Millon-Nasse Reaction

Millon-Nasse ReactionMillon’s Reagent: Mercuric ion in acidMechanism:

Tyrosine + Millon’s Reagent = complex

Millon-Nasse ReactionComplex treated with Nitrous Acid (NaNO2)

yields apink-red solution

XANTHOPROTEIC REACTION

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.

XANTHOPROTEIC REACTION

(A) NITRATED TYROSINE AND TRYPTOPHAN (B)

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.

NINHYDRIN REACTION

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

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.

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).

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.

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.

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.

It yielded a positive result for saliva thus, saliva contains proteins.

It yielded a negative result for glycine.

Saliva Glycine

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

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

Tryptophan

Formation of pinkish layer is the positive result.

Tryptophan is positive under the bromine water test

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

Samples

Saliva : +

: contains histidine and tyrosine

Histidine : +

Tyrosine : +

* Theoretical result

Saliva

HistidineTyrosine

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

POSITIVE TEST :gray : cysteine (cys)

cys

saliva

hair

* Theoretical result

Samples

Saliva : (-)

Hair : (-)

: high amount of sulfur due to cysteine

Cysteine: (+)

: sulfur was liberated

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