dr.loh protein exp (doen).docx

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AACB 3223 STRUCTURAL BIOCHEMISTRY

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FACULTY OF APPLIED SCIENCES AND COMPUTING

KUALA LUMPUR CAMPUS

DIPLOMA IN SCIENCE

(CHEMISTRY AND BIOLOGY)


LABORATORY MANUAL

Name Wong Chee Wein

ID: 13WAD01004 (AA1)

Lecturer: Dr. Loh Khye Er


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Title: Test for Proteins

Objectives:

1.

To detect the presence of peptide bond in protein by Biuret Test2. To determine the protein with Millons Test

3. To identify the effect of heavy metals salt on protein

Introduction:

Proteins are flexible and dynamic macromolecules that come in a variety of shapes and sizes, and have a

very important role in biological processes. Proteins have a function in almost all processes within cells

and the body such as cell-signaling, metabolism, the cell cycle and many more.

Protein structure (general)

Proteins have four levels of structure

The first level of the protein structure is theprimary structure which is the amino acid sequence.

The sequence of amino acids is based upon the codon sequence, a codon being a unit of three

nucleotides in the DNA or RNA sequence. In cells there are twenty common amino acids that are

used in the synthesis of proteins. Each amino acid also has a unique side chain giving it specificproperties; in addition they have an amino (N-terminus) and carboxylate (C-terminus) group

which form peptide bonds with other amino acids to form linear polypeptide chains that can

include over a hundred amino acids. A protein can consist of a single polypeptide or can be morecomplex with several different polypeptides.

The secondary structure refers to interactions between amino acids in the polypeptide chain formconformations such as alpha helices, beta strands and beta sheets (pleated sheet). These

conformations are stabilized by non-covalent hydrogen bonds between amide hydrogens and

oxygen in the amino acid backbone

The tertiary structure occurs when the secondary structure elements fold and compact. This

structure is stabilized via the non-covalent interactions of amino acids with its surrounding

environment, amino acids that are far apart in the primary structure can be brought into close

proximity.

Some proteins have a quaternary structure, which results through the interactions of two or more

of the same polypeptide or different polypeptide chain coming together to form a multi-subunit

protein..


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Peptide bonds

A peptide is a covalent bond that is formed between two molecules when the carboxyl group of one

molecule reacts with the amino group of another molecule, releasing a molecule of water. This is a

condensation reaction and usually occurs between amino acids. The resulting CO-NH bond is called a

peptide bond, and the resulting molecule is an amide.

Any number of amino acids can be joined together in chains of 50 amino acids called peptides,

50-100 amino acids called polypeptides, and over 100 amino acids called proteins. A number of

hormones, antibiotics, antitumor agents and neurotransmitters are peptides (proteins).

A peptide bond can be broken down by hydrolysis (the adding of water). The peptide bonds that

are formed within proteins have a tendency to break spontaneously when subjected to thepresence of water

Biuret Test

The Biuret reaction can be used for both qualitative and quantitative analysis of protein. The

biuret method depends on the presence of peptides bonds in proteins. When a solution of proteins

is treated with copper ions (Cu2+

) in a moderately alkaline medium, a purple colored Cu2+

-peptide complex is formed which can be measured quantitatively by spectrophotometer in the

visible region. So, biuret reagent is alkaline copper sulfate solution.

The intensity of the color produced is proportional to the number of peptide bonds that are

reacting, and therefore to the number of protein molecules present in the reaction system. The

reaction do not occur with amino acids because the absence of peptide bonds, and also that withdi-peptide because presence of only one peptide bond, but do with tri-, oligo-, and poly-peptides.

Biuret reaction needs presence of at least two peptide bonds in a molecule .The reaction occurs

with any compound containing at least two bonds of


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Material & Methods:

I. Biuret Test

3ml of soya bean extract is added into 4 different test tubes

The development of color is noted and the observation is recorded

In tabular form

3ml of 20% KOH is added in the first test tube and shake thoroughly

The steps are repeated with the substitution of distilled water and

1% of egg albumin. Any observations are recorded

1ml of 0.5% of CuSO4is added slowly drop by drop into the same

test tube


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III. Protein precipitation by Heavy Metals

3 ml of protein extract is pipetted into 4 separated test tubes

The procedure is then repeated for 3 remaining tubes using 1% of

AgNO3, 1% of MgCl2, and 1% of CuSO4

The result is then observed and recorded in a table before adding

1% of HgCl2in excess

The formation of precipitation is noted and the amount of

precipitation on a relative basis (none, very slightly, slightly, etc) is

recorded.

The steps are repeated with 2 % of egg albumin in place of the

protein extract.

The 1sttest tube is added with 2 drops of 1% HgCl2


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Result:

B. Protein Test (Biuret Test)

Test tubes Solutions Observations

1st Soya bean extract Milky brown suspension

is formed with

foam on top of

solution

2nd Distilled Water Very fade crystal

blue color solution

is formed

( negative result)

3rd 1% Egg Albumin Foam is formedon top of solution

purple violet can be

seen clearly

( positive result)

Figure 1: From left (soya bean extract), second from left, distilled water, and 1% of egg

albumin


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Millons Test

Test tubes Solutions Observation

1st

1% of egg

albumin Pink color aggregation is formed

on top of solution clumpingTogether

( positive result)

2nd Distilled water crystal clean colorless solution

with no precipitate

3rd Soya bean extract 2 layers of dry precipitate is

are formed, pinkish aggregate

at the bottom while milky white

aggregate on top of the mixture


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Protein Precipitation by Heavy Metals

Protein Extracts

Number of drops

TestTubes

ReagentSolutions 2 Drops Added in Excess

1st HgCl2 Non suspension Foamy is seen on top of solution

Milky White

solution 2 layers, very slightly cloudy on top

is formed white precipitate is formed

2nd CuSO4 Non suspension 2 Layers precipitate is formed

Milky white

solution foamy, greenish color precipitate

is formed

3rd AgNO3 No precipitate is 2 layers of precipitate is formed,

formed, a veryslight thick purplish powdery precipitate

suspension present

4th MgCl2 No precipitate is Foamy on top of solution, no visible

formed, a very

slight layers of precipitation, cloudy white

suspension solution

Figure 2: From left HgCl2, second from left, AgNO3, follow by MgCl2, and CuSO4


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2% Egg Albumin

Number of drops

Test

Tubes Solutions

2

Drops

Added in

Excess1st HgCl2 Non precipitate 2 layers of precipitate, slightly

White cloudy

white milky precipitate is

formed

suspension

2nd CuSO4 Milky white cloudy Crystal clear solution with no

suspension

precipitate, solution is slightly

crystal

blue color

3rd AgNO3 Non precipitate

2 layers of precipitate, slightly

white

White cloudymilky precipitate Ispresent

suspension

4th MgCl2 Colorless solution

Foamy on top of solution,

colorless

solution

Figure 2: From left HgCl2, second from left, AgNO3, follow by MgCl2,

and CuSO4


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Discussion:

Millons Test

Biuret Test served as a purpose of determining the properties of protein same goes to

Millons Test, in MillonsTest, it is considered as a general protein test and not a specifictest for protein, it functions as a phenolic compound detector whereby it shows positive

result when phenolic compound is present in the structure and when it is soluble, it gives a

dark red or pinkish solution.

Figure shows the structure of phenolic compound presence in protein

Biuret Test

Biuret Test, however is a much more specific test for protein, as it serves as a detector for

peptide bond, when tested positive, it will change from blue to purple violet. In this

reaction, proteins form a purple colored complex with CuSO4in a strongly alkaline solution.

When proteins and peptides (peptide bonds) treated with an alkaline solution of dilute

copper sulfate a violet color is formed. A positive test is indicated by the formation of aviolet color.

Figure shows the peptide bond formed between 2 amino acids by removal of water


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Protein Precipitation by Heavy Metals

Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt

with a protein usually leads to an insoluble metal protein salt. Heavy metal salt will

neutralize the protein. By the negative charge of protein will bind with positive charge of

metal ion. Then the protein will precipitate as insoluble metal protein salt.

Conclusion:

In this experiment, the test for 1% egg albumin by using Biuret Test shows the positive

result of purple violet solution this shows that, peptide bonds is presence in 1 % egg

albumin thats why the solutions turned purple violet color. As for Millons Test, the

experiment is also a success where it shows the presence of pinkish red aggregate on top ofthe solution,

References:

1.

http://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-

experiment.html

2.

http://shinaebiochem.blogspot.com/2013/04/practical-2-protein-

experiment.html#

3. http://www.fastbleep.com/biology-notes/40/116/780

4.

http://www.peptideguide.com/peptide-bond.html

5. http://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_fun

ctional_group_in_protein.pdf
http://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.htmlhttp://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.htmlhttp://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.htmlhttp://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.htmlhttp://shinaebiochem.blogspot.com/2013/04/practical-2-protein-experiment.htmlhttp://shinaebiochem.blogspot.com/2013/04/practical-2-protein-experiment.htmlhttp://shinaebiochem.blogspot.com/2013/04/practical-2-protein-experiment.htmlhttp://shinaebiochem.blogspot.com/2013/04/practical-2-protein-experiment.htmlhttp://www.fastbleep.com/biology-notes/40/116/780http://www.peptideguide.com/peptide-bond.htmlhttp://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_functional_group_in_protein.pdfhttp://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_functional_group_in_protein.pdfhttp://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_functional_group_in_protein.pdfhttp://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_functional_group_in_protein.pdfhttp://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_functional_group_in_protein.pdfhttp://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_functional_group_in_protein.pdfhttp://www.peptideguide.com/peptide-bond.htmlhttp://www.fastbleep.com/biology-notes/40/116/780http://shinaebiochem.blogspot.com/2013/04/practical-2-protein-experiment.htmlhttp://shinaebiochem.blogspot.com/2013/04/practical-2-protein-experiment.htmlhttp://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.htmlhttp://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.html

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