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TITLE:
Learning Characteristic and Color Reaction from Protein
B. DATE OF EXPERIMENT:
November, 28th 2014
C. PURPOSE OF EXPERIMENT:
1. Differentiate the solubility properties of protein as reversible and irraversible
2. Differentiate the denaturation reaction of protein that caused by acid, salt, and
salt from hard metal, and also the heating based on observation
3. Understand the cause of precipitation in protein identify the protein occurence
based on the color reaction
4. Identify the protein occurence based on the color reaction
D. BASIC THEORY
Protein, highly complex substance that is present in all living organisms.
Proteins are of great nutritional value and are directly involved in the chemical
processes essential for life. The importance of proteins was recognized by the
chemists in the early 19th century who coined the name for these substances from
the Greek proteios, meaning “holding first place.” Proteins are species-specific;
that is, the proteins of one species differ from those of another species. They are
also organ-specific; for instance, within a single organism, muscle proteins differ
from those of the brain and liver. (Haurowitz:2014)
Proteins are organic compounds that contain the element nitrogen as well as
carbon, hydrogen, and oxygen. Proteins are the most diverse group of biologically
important substances and are often considered to be the central compound
necessary for life. In fact, the translation from the Greek root word means “first
place.” Skin and muscles are composed of proteins; antibodies and enzymes are
proteins; some hormones are proteins; and some proteins are involved with
digestion, respiration, reproduction, and even normal vision, just to mention a
few.
Amino Acids, There are obviously many types of proteins, but they are all
made from amino acids bonded together by the dehydration synthesis. By
continually adding amino acids, called peptides, two amino acids join together to
form dipeptides; as more peptides join together, they form polypeptides. Proteins
vary in length and complexity based on the number and type of amino acids that
compose the chain. There are about 20 different amino acids, each with a different
chemical structure and characteristics; for instance, some are polar, others are
nonpolar. The final protein structure is dependent upon the amino acids that
compose it. Protein function is directly related to the structure of that protein. A
protein's specific shape determines its function. If the three-dimensional structure
of the protein is altered because of a change in the structure of the amino acids,
the protein becomes denatured and does not perform its function as expected.
Protein Structure, The three-dimensional geometry of a protein molecule is
so important to its function that four levels of structure are used to describe a
protein. The first level, or primary structure, is the linear sequence of amino acids
that creates the peptide chain.
The tertiary structure describes the overall shape of the protein. Most tertiary
structures are either globular or fibrous.
Generally, nonstructural proteins such as enzymes are globular, which means
they look spherical. The enzyme amylase is a good example of a globular protein.
Structural proteins are typically long and thin, and hence the name, fibrous.
Quaternary structures describe the protein's appearance when a protein is
composed of two or more polypeptide chains. Often the polypeptide chains will
hydrogen bond with each other in unique patterns to create the desired protein
configuration.
Characteristic of Protein,
General Characteristics of Proteins are as follows:
Proteins are organic substances, they are made up of nitrogen and also,
oxygen, carbon an d hydrogen.
Proteins are the most important biomolecules, they are the
fundamental constituent of the cytoplasm of the cell.
Proteins are the structural elements of body tissues.
Proteins are made up of amino acids.
Proteins gives heat and energy to the body and also aid in building and
repair.
Amphoteric properties of proteins due to the presence of free carboxylic
and free amino groups at the end of protein it can react with acids and
bases.
Only small amounts of proteins are stored in the body as they can be used
up quickly on demand.
Proteins are considered as the bricks, they make up bones, muscles, hair
and other parts of the body.
Proteins like enzymes are functional elements that take part in metabolic
reactions.
Antibodies, blood haemoglobin are also made of proteins.
Proteins have a molecular weight of 5 to 300 kilo-daltons.
Properties of Proteins
b. Xanthoprotein Reaction
The xanthoproteic test is a method that can be used to determine the
amount of protein soluble in a solution, using concentrated nitric acid. The
test gives a positive result in those proteins with aminoacids carrying
aromatic groups, especially in the presence of tyrosine. If the test is positive
the proof is neutralized with an alkali, turning dark yellow. This chemical
reaction is a qualitative test, determining the presence or absence of proteins.
To quantify, it is used another reaction, such as the Biuret, and an analysis is
made by photometric spectrum.
c. Ninhydrin Reaction
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect
ammonia or primary and secondary amines. When reacting with these free
amines, a deep blue or purple color known as Ruhemann's purple is produced.
Ninhydrin is most commonly used to detect fingerprints, as the terminal
amines of lysine residues in peptides and proteins sloughed off in fingerprints
react with ninhydrin. It is a white solid which is soluble in ethanol and
acetone at room temperature. Ninhydrin can be considered as the hydrate of
indane-1,2,3-trione.
d. Millon Reaction
Millon’s test is given by any compound containing a phenolic
hydroxy group. Consequently, any protein containing tyrosine will give a
positive test of a pink to dark-red colour. The Millon reagent is a solution of
mercuric and mercurous ions in nitric and nitrous acids (caution: millon’s
reagent is highly toxic and highly corrosive). The red colour is probably due
to a mercury salt of nitrated tyrosine. Millon's test is not specific for proteins
(it actually detects phenolic compounds), and so must be confirmed by other
tests for proteins such as the biuret test and the ninhydrin reaction. The
reagent is made by dissolving metallic mercury in nitric acid and diluting
with water.
e. Hopkin-Cole Reaction
The Hopkins-Cole reaction, also known as the glyoxylic acid
reaction, is a chemical test used for detecting the presence of tryptophan in
proteins. A protein solution is mixed with Hopkins Cole reagent, which
consists of glyoxylic acid. Concentrated sulfuric acid is slowly added to form
two layers. A purple ring appears between the two layers if the test is positive
for tryptophan. Nitrites, chlorates, nitrates and excess chlorides prevent the
reaction from occurring. When the violet or purple ring appears after the two
layers within an indole nucleus meet, this confirms that concentrated sulfuric
acid was added to a mixture of some sort that contained glyoxylic acid and a
protein. However, there are some products that do not show the reaction, such
as gelatin and zein.
If you have already studied the hydrolysis of amides under acidic conditions,
you will find that this is basically the same reaction. That's not surprising because
what biologists and biochemists call a peptide link (in proteins, for example) is
what chemists call an amide link. With an amide like ethanamide, the carbon-
nitrogen bond in the amide group is broken and you get a carboxylic acid formed:
Now imagine doing the same thing with a simple dipeptide made of any two
amino acids.
Instead of ammonium ions, you get positive ions made from the -NH2 groups
reacting with hydrogen ions. You need the extra hydrogen ion in the equation
(compared with the amide equation) to react with the -NH2 group on the left-hand
end of the dipeptide - the one not involved in the peptide link. If you scale this up
to a polypeptide (a protein chain), each of the peptide links will be broken in
exactly the same way. That means that you will end up with a mixture of the
amino acids that made up the protein - although in the form of their positive ions
because of the presence of the hydrogen ions from the hydrochloric acid.
5 mL protein
Precipitate
Changed Precipitate
2-3 mL protein
Precipited
Precipited
c. Denaturation because of adding formaldehyde
1-1.5 mL Formaldehide + 2 mL
Aquadest
Precipitate
3mL aquades
in test tube
Blue solution
Changing
Colour
result
2-3 ml protein solution in test
tube
result
3. Protein Precipitation
a Protein Precipitated with Ammonium Sulphate
Turbid Solution
Precipitate dissolve
b Protein Precipitated with Mineral Acid
Shake
Added CH3COOH
precipitate precipitate
Change color
b Ksanthoprotein Reaction
Yellow solution
Cooled
Added ammonia
Orange Solution
c Ninhydrin Reaction
1mL Protein
solution
d Millon Reaction
2mL Protein solution
Yellow precipitate
Cooled
Added a drop of 1%NaNO2 solution
Heated
Precipitate change
Mixed solution
Form 2 layers
5. Hydrolisis protein and suphur test
1 mL protein solution in
test tube
Pb-acetate
precipitate
G. TABLE RESULT OF EXPERIMENT
1. Denaturation Protein
a. Denaturation because of adding acetic acid Before :
Milk : white Protein in milk and white egg will Protein
5 mL protein Egg protein : denaturate because adding acid which sign denaturized
colorless, thick to form precipitate/ flake because add acid
CH3COOH: colorless
in the test tube
Added 2 drops acetic acid 1N
shake After :
I egg protein
Added CH3COOH:
Precipitate colourless + white
precipitate+ heated :
Heated the tube in the steam bath white solid
water for 5 minutes
Observed the change of precipitate II milk protein
Added CH3COOH:
Changed Precipitate colourless + white
solution+ heated :
white solid
2-3 mL protein
Before : Protein in milk and whte egg will white egg
Milk : white color denaturate if heated denaturate after
Egg protein: heat
colorless
Ammonium sulfat
sulfat: colorless
After :
Milk :
milk+heated: white
precipitate
coagulate,
cooled: liquid
Tube I:
Added ammonium
sulfate: white
solution
heated: white
coagulate (++)
Tube II :
heated: white
coagulate (+)
Egg protein :
Egg protein+heated:
white precipitate
coagulate
cooled: liquid
Tube I:
Added ammonium
sulfate: colorless
Heated : white
coagulate (++)
Tube II :
heated: white
coagulate (+)
After:
Added 2-3mL protein solution
Aquadest + HCl :
Note the changing color
colorless
Added kongo orange
Changing Colour paper in pH 2
Milk:
Added milk protein:
White (aq)
Paper : orange pH 3
Egg:
Added Egg protein:
Colourless (aq)
Paper : orange pH 3
Before :
3mL dilute NaOH solution in NaOH:colorless
test tube Pp indicator :
colorless
Added 2 drops of pp indicator After:
Milk protein + NaOH
result
: white solution
2-3mL protein Added PP:pink
solution in test solution
tube Egg protein + NaOH
Added a drop of PP indicator : white solution
Added NaOH solution Added PP : pink
solution
Result
After:
Milk protein +
NaOH : white
solution
Added PP: pink
solution
Egg protein +
NaOH :white
solution
Added PP : pink
solution
3 Protein precipitation
a. Protein precipitate with Ammonium Before : No chemical reaction so the precipitate Precipitation at
Sulphate Milk protein : white easy to dissolved. protein in milk &
color white egg with
3-4mL protein solution
in test tube Egg : colorless ammonium
Ammonium sulphate is
Added 3-4mL saturated sulphate: colorless reversible
ammonium sulphate solution Aquades : colorless precipitate.
Shake the tube slowly
After :
Turbid Solution
Egg+(NH4)2SO4
Taken 1mL turbid protein saturated:turbid
solution into test tube solution + aquades
: colorless
Added 2-3mL aquades
precipitate dissolve
Shake the solution
Precipitate Milk+(NH4)2SO4
dissolve saturated: turbid
white + aquades:
turbid white
solution(--)
b. Protein precipitate with mineral acid Before: Forming salt substance from acid Precipiate has
Milk : white reaction with amin group in protein irreversible
1mL HNO3 1-2mL HCl saturated
Egg protein : gotten precipitate Precipiate has
saturated
colorless Forming salt substance from acid reversible
HCl : colorless reaction with amin group in protein and
Put into test tube added 1-15 HNO3: colorless gotten precipitate which easy to dissolve
mL protein solution drop in water
by drops from partition After:
tube milk
Put the test tube vertically HNO3+ milk: white
ring+ shake: yellow
turbid solution+
White ring formed HCl: show white
precipitate
Shake Egg
Added CH3COOH Added HNO3: white
ring (++)+ shake:
yellow turbid
Get more The solution solution+ HCl+ egg
precipitate more colourless solution : white
precipitation+
shake: white turbid
solution
c. Protein Precipitate by Heavy Metals
Solution
1mL protein
solution
Put into test tube
Added drop y drop PbSO4 Egg protein + PbSO4:
Shake
blue solution +
shaked: precipitated:
precipitate Added PbSO4 : blue
solution
Added PbSO4
until dissolve
solution
4 Reaction of Protein Color
a. Biuret Reaction
Tube I Biuret reaction is color reaction which is Protein in milk
3mL protein solution + Egg peptida group (-CO-N) and protein positif and white egg
Added 1 mL 40% NaOH protein+NaOH: reaction with form complex substance contain peptida
colourless+ CuSO4 : with Cu 2+ and N foarm peptida bonding group which sign
purple turbid molecule. with purple
Put into test tube Tube II colour
Added drop by drop 0.5%
Milk +NaOH: white
CuSO4 solution
solution+ CuSO4:
purple
Change color
b. Xanthoprotein Reaction
Before: Xanthoprotein reaction can be done
There is amino acid
3mL protein solution Concentrate HNO3: because nitrasi reaction at benzenaring
in the protein with
colorless from amino acid in protein. test called
the orange colour
NH3 : colorless positive if the colour become orange
Added 1mL concentrate
HNO3 After :
Heated the mixture
Milk protein+ HNO3
+ heated : yellow
turbid, +
Yellow solution
NH3:orange turbid
Egg protein+ HNO3 +
Cooled the solution heated: yellow
Added ammonia until the turbid + NH3 :
color change into orange orange turbid
Orange
Solution
c. Ninhydrin Reaction
1mL Protein solution Before: Nynhydrin + protein become complex Milk and egg
Ninhydrin :colorless reaction become positif
Put into test tube
Added 10 dros ninhydrin result to
Heated 10 minutes After : nynhydrin test
Milk protein + because the color
nynhydrin + heated: dark purple from
dark purple turbid milk and egg.
Color
change Egg protein +
nynhydrin + heated
: dark purple turbid
d. Millon Reaction Before: Foming red precipitate because the egg Protein in milk
Hg2SO4: colourless protein & milk protein contain of and egg contain
2mL Protein tirosin/tryptofan which is reacted with Hg amino acid tirosin
solution After: from millon reactant. and triptofan
Tube I
Added 1 mL millon reagent
Heated the mixture Milk+egg + millon: 2
phase form
Heated
Yellow precipitate
Milk : orange
precipitate
Cooled Egg : orange
Added a drop of 1%NaNO2 solution precipitate
Heated the precipitate
NaNO3adding +
heated :
Change
Milk : red
color
precipitation
Egg : red
precipitation
e. Hopkin Cole Reaction Before : The reaction is positive if form purple The result is
Milk: white solution ring because form condensation in zone positive because
1mL Protein Egg:colorless indole and triptofan with aldehide. there is purple in
solution solution middle of solution
Formaldehyde: / ring
Put in the test tube olorless solution
Added 1 drop formaldehyde Millon reagent :
Added 1 drop HgSO4 colorless solution
H2SO4 : colorless
Mixed solution solution
Before : The reaction positive if form purple ring Protein from egg
Milk : white(++) because form condentation in zone indole and milk positive
1 mL protein solution in Egg solution: and triptofan with aldehide contain of Sulphur
test tube colorless Protein which contain of amino acid with , marked by the
NaOH 40%: colorless S atom will given black color if reacted formation of black
Added 1mL 40% NaOH solution Pb-acetic: colorless with Pb-acetate formed PbS. precipitate (PbS)
Heated the mixture for a minutes
Added a drop of Pb-acetate After Tube I
Milk+NaOH40%:
turbid white Pb(CH3COO)2 (aq) + 2S2- PbS (s) +
Pb-acetate solution 2CH3COO- (aq)
precipitate Milk+NaOH
40%+heated : turbid
yellow
Milk+NaOH
40%+heated+Pb-
acetic: black
precipitate
After Tube II
Egg solution+NaOH
40%:colorless
Egg solution+ NaOH
40%+heated:
colorless
Egg solution+NaOH
40%+heated+Pb-
acetic: black
precipitate
H. ANALYSIS
1. Denaturation of Protein
a. Denaturation Because of Adding Acid
From the experiment that we did, can known that protein in egg
probably contain of albumin and protein in milk probably contain of casein
and triptofan did the denaturation which is cause the forming of precipitate
of protein with the adding of acetate acid, because protein did the structure
changing from functional structure of protein. Denaturation of protein will
be bigger happen by heating. This caused that protein did the separation of
non-covalent bonding because of the increasing of temperature. So the
precipitate which is produced is increase.
b. Denaturation Because of Heating
From the experiment that we did, it can be known that heating wil
denaturate the protein that signed with the white precipitate which is
caused of the sparating of non-covalent bonding and the decreasing of the
ability to bond the water in protein so that the protein was clot and form
precipitate. Tha adding of ammonium sulphate as a denaturation agent.
c. Denaturation Because of the Adding of Formaldehyde
In this experiment, the adding of formaldehyde will denaturate
protein because of the forming of derivate of dimethyl amino acid because
of the reaction between formaldehyde with amino group in protein. The
amount of the precipitate shows the quantity of protein which is
denaturated. The precipitate from egg is more than milk. From that three
experiment that we did about the denaturation of protein, known that
protein will denaturation or destroying with some did like the adding of
acid, heating, and the adding of organnic compound. The break of protein
can be identified with the precipitate produced.
2. The Amphoter Characteristic of Protein
To know that protein have amphoter characteristic, this experiment is
did in acid and base condition. This, the goal of the addition of acid (HCl) is
to know that protein can do as base and also acid showed by the changing
color into turbid white (for milk) and colorless (for egg) after the addition of
protein solution and also by the changing color into pink solution (for milk)
and clear pink (for egg) after the addition of PP indicator. This shows that
protein have amphoter characteristic i.e acid and base.
3. Protein Precipitation
a. Precipitation of Protein by Ammonium Sulphate
Egg
Milk
Egg
Milk
While in egg solution did the same do and produced the clear
purple solution. The purple color which is produced in egg protein is more
saturated than the milk protein. This show that the protein solution in egg
have more peptide bonding or longer than the peptide bonding in milk.
b. Xantoprotein Reaction
Egg
Milk
F
r
om this experiment can be identified that between egg protein and milk
give the positive reaction contain of amino acid with benzene core like
phenylalanine, tyrosine, albumin, tryptophan, etc which is signed by the
forming of yellow precipitate.
c. Ninhidrine Reaction
Egg
At this ninhidrin reaction test did with set the pH in egg protein
solution which is 8 before, becomes 6. Then, added 10 drops of ninhidrin
reagent solution 0,2%. After heated for 10 minutes, it form dark purple
turbid solution. The dark purple turbid solution produced shows the
positive ninhidrin test, because at amino acid there’s carboxyl group react
with NH3, with the process of decarboxilation produce an amina. Amino
group in amino acid can react with nitrite acid and release the nitrogen gas.
Amino acid, ammonia and primer amino group in protein if boiled with
protein solution at pH 6 and with the ninhidrin and also hidrindatin makes
the solution becomes purple.
Milk
d. Millon Reaction
Egg
Milk
Egg
Milk
Egg
Milk
Answered:
Hydrogen bonding
This bond is formed between NH or OH group and C=O in peptide
bonding or COO in R group.
J. CONCLUSIONS
REFERENCES
1. Protein Denaturation
Put 5 ml egg protein into test Put 5 ml milk protein into Each tube was added with
tube 1 test tube 2 CH3COOH
Formed white precipitate in Then heated in steam bath Egg protein become white
egg protein solution for 5 minutes solid (coagulate)
Milk protein become solid
(coagulate)
b. Denaturation by heating
Put 3 ml egg protein (tube 1) Then heated in steam bath Egg protein (tube 1) divided
and 3 ml milk protein (tube for 1 minutes into 2 tubes
2) Become white solid
precipitate
Milk protein (tube 2) divided Tube 1 of each protein Then tube 1 and 2 of each
into two tubes solution added (NH4)2SO4 protein are heated
NaOH added PP indicator : 3 ml protein added NaOH Each tube was added with
pink solution and PP : milk (pink turbid) CH3COOH
Egg (pink clear solution)
3. Protein Precipitation
a. By adding ammonium Sulfate
Put 4 ml egg protein (tube Then each tube added 3- Then added aquades egg
1) and milk protein (tube 2) 4 ml (NH4)2SO4 saturated protein = colorless, milk
protein : white turbid
b. Adding by mineral acid
HNO3 + egg protein (yellow) HNO3 + milk protein Milk and egg protein then
and HCl + egg protein (yellow) and HCl + milk added NH3 become more
(white) protein (white) yellow turbid solution
Milk and egg protein added Then heated in steam bath Egg protein become white
by CuSO4 (tosca solution) for 5 minutes solid (coagulate)
A. Protein Color Reaction
a. Biurette reaction
Put 5 ml milk and egg Both of tube added NaOH Both of tube added CuSO4,
protein solution into test the milk protein solution
tube become turbid purple and
egg protein become clear
purple
b. Xanthoprotein reaction
Putted 3 ml milk and egg Both of tube added HNO3 Then added NH3 become
protein solution into test and heated become yellow orange
tube turbid
c. Ninhydrin Reaction
Putted 3 ml milk and egg Both of tube added Then the tube heated in
protein solution into test ninhydrin steam bath
tube
Putted 2 ml milk and egg Both of tube added Hg2SO4 Then the tube heated in
protein solution into test steam bath
tube
Putted 2 ml milk and egg 1 ml protein egg solution Added 1 drop formaldehyde
protein solution into test
tube