Protein Lab Report 3

7/24/2019 Protein Lab Report 3

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Introduction

Proteins are organic compound that are arranged in a linear chain. The word

protein refers to a type of molecule in food that can be broken down into amino

acids.

The body needs twenty amino acids to function as a biological machine and it can

synthesize eleven of these by itself. However there are nine, called essential amino

acids that the body cannot create and has to gain through the consumption of

food. hen we eat, the body breaks down the protein in food in order to create the

amino acids that it needs. Proteins are large biomolecules, or macromolecules,

consisting of one or more long chains of amino acid residues. Proteins perform a

vast array of functions within living organisms, including catalyzing metabolic

reactions, !"# replication, responding to stimuli, and transporting molecules from

one location to another. Proteins di$er from one another primarily in their se%uence

of amino acids, which is dictated by the nucleotide se%uence of their genes, and

which usually results in protein folding into a speci&c three'dimensional structurethat determines its activity. (ikipedia)

#mino acids are biologically important organic compounds containing amine ('"H*)

and carbo+ylic acid ('--H) functional groups, usually along with a side'chain

speci&c to each amino acid. The properties of the side chain are what determines

the classi&cation of the amino acid into weak acid, weak base, hydrophilic and

hydrophobic. (ikipedia)

The main obective of this e+periment is to study the general reactions of proteins

and to study the / groups of the proteins. These obectives are achieved by carrying

out several tests such as 0iuret test, "inhydrin test, 1anthoproteic Test, 2illons testand 3akaguchis test. (0iochem practical 2anual)


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Results

Biuret Test

No Sample Observation Interpretation1 0ovine serum

albumin

The solution turns from blue

to dark purple/violet

Protein is

present. It

conrms the

presence of

peptide bonds in

the protein! asein The solution turns from blue

to li"ht purple/violet

Protein is

present. It

conrms thepresence of

peptide bonds in

the protein# 4lycine No chan"es. The solution

remains in blue colour

Protein is absent

. $l%cine is an

amino acid. It

conrms the

absence of

peptide bonds& 5rea No chan"es. The solution

remains in blue colour.

Protein is

absent. 'rea is

result of the

breakdo(n of

amino acid.

)ence the

peptide bonds

are absent* H*6(water) No chan"es. The solution

remains in blue colour.

Protein is

absent. +ater

acts as a

ne"ative controlof the

e,periment.

Peptide bonds

are absent.

Ninh%drin Test


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No Sample Observation Interpretation1 4lycine - violet blue solution is

formed

ree amino acid

"roup is present

in "l%cine.! Proline ark %ello( colour solution

(ith a (hite precipitate isformed.

Proline is an

imino acid. It is asecondar% amino

acid and it has a

rin" structure.

)ence the free

amino acid "roup

is absent.# 0ovine serum

albumin

- clear solution is formed

0anthoproteic Test

No Sample Observation Interpretation1 4lycine olourless solution is formed The ben2ene rin"

is absent. Since

"l%cine is

aliphatic aminoacid it do not

possess a

ben2ene rin".! Phenylalanine Pale %ello( solution is formed Phen%lalanine is

an aromatic

amino acid. It

has a ben2ene

structure .# 0ovine serum

albumin

Oran"e red solution is formed This conrms

thatbovine

serum albumin is

an amino acid

containin" a

ben2ene rin"

3illon4s test


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No Sample Observation Interpretation1 4lycine No chan"es are observed.

lear solution is formed

Protein is absent

since "l%cine is

an amino acid! 0ovine serum

albumin

ark reddish pink precipitate

is formed

Protein is

present# asein 5i"ht reddish pink precipitate

is formed

Protein is

present

Saka"uchi4s test

No Sample Observation Interpretation1 4lycine 5i"ht %ello(

solution is

formed

-r"inine6a t%pe

of amino acid7 is

absent! #rginine 5i"ht reddish

pink solution is

formed

-r"inine is

present

# 5rea 5i"ht %ello(

solution is

formed

-r"inine is

absent

iscussion

Proteins are organic compounds made of amino acids.The amino acids which is the

building block of proteins oined together by the peptide bonds. 7n a normal functional

protein, the long chain of amino acids is folded into a 8' dimensional shape. 9very

protein has a uni%ue 8'dimensional shape that is suited to its biological function in a

living organism. The unfolding of the 8! shape will denature the protein where the

protein will no longer be able to carry out its biological function and will usually be

destroyed by the cell.

There are several %uantitative tests for determining whether amino acids or proteins

are present in solution. These tests are speci&c for the presence of peptide

bonds,certain types of side chains and the type of secondary structure present.


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The biuret test is a chemical test used for detecting the presence of peptide bonds.

7n the presence of peptides, a copper(77) ion forms violet'colored coordination

comple+es in an alkaline solution. The 0iuret reagent contains copper ions which

give it a blue color. The copper ions will interact with a compound that contains two

or more peptide bonds, resulting in the formation of a violet:purple'colored product.

The dipeptide bonds in proteins react with the u*; ions to form the comple+. Thelone pairs present on the "itrogen of the peptide linkage bonds with the cupric ions

in the reagent to create a violet purple colour change. hen a compound does not

have at least two peptide bonds, it will not react with the 0iuret reagent, and no

purple color will appear . The solution will remain a shade of blue due to the copper

ions. 0iuret test is speci&c for testing Proteins, 7t helps to di$erentiate between

Proteins (;ve) and #mino #cids ('ve).

7n the e+periment above, the 03# forms the highest intensity of the purple comple+

compared to caseine because of the number of peptide bonds in 03# is higher than

in caseine. The number of peptide bonds present in the protein is proportional to the

intensity of the violet coloured comple+ formed. Thus, the biuret reaction is the

basis for a simple and rapid colorimetric reagent of the same name for

%uantitatively determining total protein concentration The violet coloured comple+

formed con&rms the presence of proteins, so in 03# and caseine proteins are

present. hile glycine shows negative result in the 0iuret test because glycine is

the simplest amino acid. # peptide bond (amide bond) is a covalent chemical bond

formed between two amino acid molecules. 3ince there are no peptide bonds

present in the glycine alone, it shows a negative result in the 0iuret test. 5rea which

is the breakdown of amino acids shows a negative result in 0iuret test since the

peptide bonds are absent .0ut when the urea is heated it may give a positive result

because the heated urea forms the biuret compound that reacts with the cupric ionspresent in the reagent to form the coloured comple+.

#lthough amino acids are the building block of amino acids, but it give negative

results in 0iuret test because atleast T- or more peptide bonds are re%uired for

the formation of the chelate comple+. 7n single amino acids there are no peptide

bonds present hile in dipeptides only -"9 peptide bond present hence this gives

a negative result.

The biuret test can be e+tended to %uantitatively measure the concentration of total

protein using absorption spectroscopy method. 5sing a spectrophotometer which

follows the 0eer'


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"inhydrine test utilized to identify amines in particular alpha amino acids present in

the solution. 7t is commonly used to detect the lysine in &ngerprints. "inhydrin (*,*'

!ihydro+yindane'=,8'dione) is a chemical used to detect ammonia or primary and

secondary amines. hen reacting with these free amines, a deep blue or purple

color known as /uhemann>s purple is produced. The ninhydrin reagent will react

speci&cally with a primary amino functional group on a compound, resulting in theformation of a violet:purple'colored product. hen a compound doesnot have a

primary amino group, it will not react with the ninhydrin reagent, and no purple

color will appear (solution will remain colorless)

7n the e+periment above , 03# shows positive result by forming violet blue colour

solution. The alpha amino group of the free amino acid react with ninhydrin to form

a blue to purple colour change. However, some amino acids such as the imminoacid

proline react with ninhydrin di$erently to form a bright' yellow colour change. This

is because proline has a ring structure as shown in the diagram below.

The blueish'purple result is usually associated with primary amino acids. 7n these

amino acids, the " is free to react with ninhydrin. However, in proline, the " is not

available for reaction as it is locked in the ring structure. Therefore no ammonia is

produced, so no blue color is presented.

The obective of +anthoproteic test is to di$erentiate between aromatic amino acids

which give positive results and other amino acids which do not possess the benzene

ring.#mino acids containing an aromatic nucleus form yellow nitro derivatives on

heating with concentrated H"-8. The salts of these derivatives are orange in color.

oncentrated nitric acid reacts with the aromatic rings that are derivatives of

benzene giving the characteristic nitration reaction. 03# contain activated benzene


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rings which are easily nitrated to orange colored compounds upon heating . The

aromatic ring of phenylalanine dose not react with nitric acid despite it contains a

benzene ring, but it is not activated, therefore it will not react.

2illons test is speci&c for tyrosine, the only amino acid containing a phenol group, a

hydro+yl group attached to benzene ring.The main obective of this test is to detect

the presence of tyrosine in the sample. 7n 2illon>s test, the phenol group of tyrosine

is &rst nitrated by nitric acid in the test solution.Then the nitrated tyrosine

comple+es mercury ions in the solution to form a brick'red solution or precipitate of

nitrated tyrosine, in all cases, appearance of red color is positive test which

indicates the presence of proteins. 2illons test is given by any compound

containing a phenolic hydro+y group. onse%uently, any protein containing tyrosine

will give a positive test of a pink to dark'red colour.However ,2illon>s test is not

speci&c for proteins (it detects phenolic compounds), and so must be con&rmed by

other tests for proteins such as the biuret test and the ninhydrin reaction.

7n the e+periment above, glycine shows negative results since it is an amino acid.

#nd it does not possess phenolic hydro+yl molecule attached to it. hile 03# and

caseine give positive result which is con&rmed by the formation of dark red

precipitate. This indicates that protein is present.

3akaguchi test is a speci&c %ualitative test for the detection of amino acid

containing gauanidium group ?/'"H'@ ("H*)*; '"H*A. 7n other words its a test for

guanidines, for an e+ample arginine. 7n alkaline solution, arginine react with B'

naphthol and sodium hypobromite :chlorite as an o+idizing agent, to form red

comple+es as a positive result.7n the e+periment above only in arginine thelightreddish pink solution is formed which indicates the positive result.

onclusion

7n conclusion we can state that there are several e+perimental analysis that can be

done to con&rm the presence of protein and also to study the side chain, / group

and structure of the protein. 0ased on all the reactions above, di$erent protein and

amino acids shows di$erent characteristic di$erence. Hence di$erent tests must bedone to con&rm the speci&city of the proteins. 0y the end of this e+periment the

obectives of this e+periment were met.

Protein Lab Report 3

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