biochem ppt

7/15/2019 Biochem Ppt

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EXPERIMENT 6Qualitative Analysis of Proteinsand Amino Acids

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Objectives

To identify the aminoacid present in aprotein sample basedon its color reaction

with specific reagents.

Qualitatively analyze

amino acids andproteins




FLOW DIAGRAM




1. BIIURET TEST

Observecolor Mix

+ 5 dropsCuSO4 solution

1 mlNaOH

+

2 ml

peptone

+




2.NINHYDRIN TEST

describe color

heat to boiling in water bath

2 ml peptone + 1 ml Ninhydrin reagent




3.XANTHOPROTEIC TEST1 ml peptone

+

5 dropsconc.HNO3

mix heat towater bath

heat for another 1

minute

cool under tapH2O

add few dropsconc. NH4OH

note thecolor




4.MILLON’S TEST

1 ml peptone +

4 drops Millon’s reagent

boil in water

bath for 10minutes

cool in runningwater

add 4 dropsNaNO2

observe the

color of precipitate




5.HOPKIN’S COLE TEST

1ml peptone

+ 2 dropsHopkin’s

Colereagent

Mix

Add 1 ml

conc. H2SO4 along theside of

inclined testtube

Note the

color of the junction

between 2layers




6.SAKAGUCHI TEST

3 mlpeptone +

+ 2 dropsNaOH

solution

+ 4 dropsα

naphtholsolution

+ 10drops

brominewater

Observethe color




7.LEAD ACETATE TEST

1 ml peptone +5 drops NaOH

solution

boil in water bath for 3minutes

Cool

add 3 dropslead acetatewith mixing

color of precipitate




Results and DataBiuret Test – green

Ninhydrin Test – Blue

Xanthoproteic Test – Light Yellow




Millon’s Test – Light Brown

Hopkin’s Cole Test- Yellow

Sakaguchi Test- Brown

Lead Acetate Test- White




LEARNINGS and VALUES:

Values

-teamwork-patience

-discipline

Proteins are complex, high molecular weight

biomolecules composed of amino acids joinedtogether by peptide bonds. The twenty aminoacids normally found in proteins differ intheir amino groups in the side chains. These

respond to specific qualitative tests withcertain chemical compounds chemicalreagents and thus become the basis of theirdetection whether as free amino acids or in

combined form as in peptides or proteins





A peptide or a peptone is a shortchain of amino acid residues. A polypeptide is a long-chain of

amino acid residues. A proteinconsists of one or morepolypeptides which are folded in aglobular or fibrous form, so that it

performs a biological function. A peptone is a secondary derivedprotein form by hydrolysis of proteoses. It is not heat coagulableprotein. Peptone contains traces of cysteine and cystine amino acid.




BIURET TEST A biuret test is a chemical test used for detecting the

presence of peptide bonds. It works by copper sulphateions reacting with the peptide bonds of proteins toproduce a pink to purple color in an alkaline solution.Comparison of colour is made from a standard solution

whose protein concentration is known.




NINHYDRIN TESTNinhydrin test is the general test given by all the

amino acids. One can identify the amino acids by performing this test. The amino group of amino acidsreacts with the ninhydrin an oxidizing agent and theamino acid gets itself oxidative decarboxylated anddelaminated to an aldehyde.

It reacts with α amino groups of protein and freeamino acid to give a blue or bluish purple complex(Ruhemann’s purple).




Xanthoproteic Test (Mulder’s test)

This test gives positiveresult with aromatic aminoacids containing phenylgroups in their structures (

phenyl alanine, tyrosine &tryptophan).Peptone givesa yellow color on this test.

Xanthoprotein is a yellowacid substance formed bythe action of hot nitric acidon albuminous or protein

matter and is changed toa deep orange-yellowcolour by the addition of ammonia.




Principle:

This test is based on the

ability of aromatic aminoacids containingsubstituted phenyl groups

to react with concentratednitric acid to give dinitroderivates. These are furtherconverted to quinoid

structures by reacting withstrong alkali.




Millon’s testIt is generally Known as Millon-

Nasse’s Test. This test gives apositive result with tyrosineamino acid it gives a positiveresult both in free state as well as

being present as constitutive of protein.

Peptone gives a pinkish

red color due to thepresence of tyrosine.




PRINCIPLE:

Phenyl /radical or hydroxy

benzene radical present intyrosine reacts with millon’sreagent to yield a purple rednitro-hydroxy phenyl

mercurial, on heating withmercuric sulfate and sodiumnitrite. This reaction is alsogiven by phenolic compound.

In amino acids only tyrosineand its derestatives gives thepositive result.




Glyoxylic acid test (Hopkin-cole’ test):

This test is given by those amino acids which

have the indole groups.The indole groups present in the tryptophanmolecules react with glyoxylic acid inpresence of con. H2SO4 forms a condensation

product which is reddish violet, the sulfuricacid acts as dehydrationg agent, eliminating amolecule of water. Tryptophan glyoxyliccondensation product ( reddish violet).

There’s a violet ring at the junction of twosolutions due to the presence of tryptophan.




SAKAGUCHI TESTPRINCIPLE : The amino acid containingguanidino group reacts with alpha- Naphtholand on oxidizing agent like bromine water togive red colour product. This test is positivefor arginine.

In a test for proteins, the guanido group is inthe basic amino acid Arginine. The guanido(NH2 – C = NH) group of free arginine orarginine residues in the protein reacts α

naphthol and sodium hypobromite(bromine water and NaOH) to give a bright red coloredcomplex.

Peptone gives bright red color due to the

presnce of arginine.





LEAD ACETATE TEST

When proteins containing cysteine orcystine residues are boiled with strongalkali,organinc sulphur is converted tosodium sulphide. The sodium sulphide

reacts with lead acetate to form leadsulphide precipitate (insoluble) whichis black or brown in color.

Peptone gives light black or brown

color because it traces the presence of cysteine and cystine.




APPLICATION:

Amino Acid Disorders Screening

Amino acid disorder screeningchecks for inherited disorders inamino acid metabolism. Tests aremost commonly done on newborns.Two tests are available, one using ablood sample and the other a urine

sample.




Urine TestProtein in the urine is a warning sign. It

may indicate kidney damage or disease orit may be a transient elevation due to aninfection, medication, vigorous exercise,or emotional or physical stress.

When kidney damage is present, theamount of protein present is generally associated with the severity of damage,

and increasing amounts of protein overtime indicate increasing damage anddecreasing kidney function. Proteinuria isassociated with many diseases and

conditions




REFERENCE:http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html

http://books.google.com.ph/books?id=nPphGdHO_XcC&pg=PA22&lpg=PA22&dq=result+of+peptone+in+ninhydrin+test&source=bl&ots=n_NLKO5DFz&sig=r3KshRZQ6Y7sBogFjXlV6FMQW7s&hl=en&sa=X&ei=EQMVUfmMHaiXiQfkmoHgDw&ved=0CDMQ6AEwAQ#v=onepage&q=result%20of%20peptone%20in%20ninhydrin%20test&f=false

http://www.angrau.ac.in/media/10940/bicm101.pdf

http://elearning.najah.edu/OldData/docs/51284_Protein.pdf http://ask.reference.com/related/Ninhydrin+Reaction+with+Amino+A cids?qsrc=2892&l=dir&o=10601

http://altmedicine.about.com/od/healthconditionsatod/a/amino_acids.htm

http://www.google.com.ph/books?hl=en&lr=&id=nPphGdHO_XcC&oi=fnd&pg=PA1&dq=color+of+peptone+in+sakaguchi+test&ots=n_NLONbCGt&sig=_TFkTLuECLoKx_8ZVO-m7UUlfWY&redir_esc=y#v=onepage&q&f=false

http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html

http://books.google.com.ph/books?id=nPphGdHO_XcC&pg=PA22&lpg=PA22&dq=result+of+peptone+in+ninhydrin+test&source=bl&ots=n_NLKO5DFz&sig=r3KshRZQ6Y7sBogFjXlV6FMQW7s&hl=en&sa=X&ei=EQMVUfmMHaiXiQfkmoHgDw&ved=0CDMQ6AEwAQ






http://elearning.najah.edu/OldData/docs/51284_Protein.pdf

http://ask.reference.com/related/Ninhydrin+Reaction+with+Amino+Acids?qsrc=2892&l=dir&o=10601




http://www.google.com.ph/books?hl=en&lr=&id=nPphGdHO_XcC&oi=fnd&pg=PA1&dq=color+of+peptone+in+sakaguchi+test&ots=n_NLONbCGt&sig=_TFkTLuECLoKx_8ZVO-m7UUlfWY&redir_esc=y













http://elearning.najah.edu/OldData/docs/51284_Protein.pdf















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