Qualitative Tests Proteins & Amino Acids in Saliva

Co. Cordero. Cruz. De Jesus

Sakaguchi Test
Test for the presence of guanidine

Sakaguchi Test
Test for the presence of guanidine

ARGININE

Sakaguchi Test
Reagents: -naphthol NaOH *NaOCl Mechanism:

+ NaOH, pH

zwitterionic form

Sakaguchi Test

condensation reaction with

red/wine-colored solution

Millon-Nasse Reaction
For compounds containing a phenolic hydroxy group Amino acid: Tyrosine
Compound must be

validated as protein/ amino acid to confirm presence of tyrosine

Millon-Nasse Reaction
Millons Reagent: Mercuric ion in acid
Mechanism: Tyrosine + Millons Reagent = complex

Millon-Nasse Reaction
Complex treated with Nitrous Acid (NaNO2) yields a pink-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 REACTION
Using 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 REACTION
Triketohydrindene 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 purplecolored compund

NINHYDRIN REACTION
A 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 REACTION
Ninhydrin 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 REACTION
-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 Test
Presence 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 test

The 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 Histidine

Tyrosine

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)

hair cys saliva

Samples
Saliva : (-)

Hair : (-)
: high amount of sulfur due to cysteine Cysteine: (+) : sulfur was liberated
* Theoretical result

References
n.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/D iazotization%20related%20reactions.gif n.a.(n.d.). Hair fibers. Date Retrieved August 3,2010,from http://www.keratin.com/aa/aa012.shtml