HI GUYS CHECK THIS OUT OMG KJASDLKFJDSLF THIS WAS NUNG 2016 LANG AND

UST PA SILA SO
https://www.scribd.com/document/307023652/BioOrganic-Chemistry-Experiment-8-Chara
cterization-of-Proteins-and-Amino-Acids
^^ omg niceee hehe

http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1

LETS INCLUDE ESSENCE OF KNOWING THESE IN THE PRACTICE IF KAYA

A. PROTEIN DENATURATION
1. Define denaturation
Denaturation is the process of modifying the molecular structure or shape of a protein
through some form of external stress like heating, applying alkali or acid, and vigorous shaking.
It involves the breaking of weak linkages or bonds within a protein molecule that are responsible
for the highly ordered structure of the protein in its natural state. Proteins that are denatured are
mostly insoluble. It also has a more loose and incoherent structure.
2. What physical and chemical agents are capable of denaturing proteins? Give the type of
bonds or attractive interactions disrupted by these denaturing agents.
3. What concentration of alcohol is most effective as a disinfectant? Why?
70% IPA solutions penetrate the cell wall more completely which permeates the entire
cell, coagulates all proteins, and therefore the microorganism dies. ​Ethyl alcohol kills bacteria
mainly through 2 mechanisms: protein denaturation and dissolving the lipid membrane.
Proteins, the machinery of the cell, must be dissolved in water in order to properly function. ​70%
percent of alcohol is ideal to a stronger solution. Pure alcohol coagulates protein in contact.
Suppose the pure alcohol is poured over a single celled organism. The alcohol will go through
the cell wall of the organism in all direction, coagulating the protein just inside the cell wall. The
ring of the coagulated protein would then stop the alcohol from penetrating farther from the cell,
and no more coagulation would take place. At this time the cell would become inactive but not
dead. Under the favorable conditions the cell would then begin to function. If 70 percent of
alcohol is poured to a single celled organism, the diluted alcohol also coagulates the protein, but
at a slower rate, so that it penetrates all the way through the cell before coagulation can block it.
Then the entire cell is coagulated and the organism dies.
4. Explain how protein denaturation using heat, alcohol, and heavy metal ions is used in the
medical field.
Heating​ to denature proteins are used to sterilize medical supplies and instruments thus
destroying the bacteria. This occurs because the heat increases the kinetic energy which
triggers the molecules to vibrate rapidly and violently, and causes the bonds to disrupt.
A ​70% alcohol​ can penetrate the bacterial cell wall and denature the proteins and
enzymes inside of the cell which can be used as a disinfectant on the skin. It is important in the
medical field because it can help in preventing the spread of bacteria.
Protein denaturation using ​heavy metal ions
 5. What amino acids in a protein are reactive with heavy metal ions? Explain and illustrate
with an equation.

B. COLOR REACTIONS
1. Xanthoproteic Reaction
a. Give the principle involved in this test. Illustrate with an equation.
Concentrated nitric acid reacts with an aromatic group present in the amino acid side
chain that will lead to a nitration reaction which will give off a solution with a
yellow-orange color.
*For phenylalanine, it will be negative with the Xanthoproteic test due to the
unactivated/deactivated aromatic group in the amino acid. Also, the phenyl group is
very stable, thus not reacting with the nitric acid.

This picture illustrates the reaction of nitration of ​Tyrosine​ as an example of a Xanthoproteic

Reaction

b. What group in an amino acid is identified by this test?

Aromatic group.
c. Name the amino acids that will be positive with this test.
Phenylalanine, Tryptophan, Tyrosine
 d. What is the isoelectric point? Explain how pH changes in a protein solution affect its
solubility.
- The surface of a protein has a net charge that depends on the number and
identities of the charged amino acids, and on pH. At a specific pH the positive
and negative charges will balance and the net charge will be zero. This is called
the isoelectric point.
- Occurs around pH 5.5 to 8.
- A protein has its lower solubility at its isoelectric point.
- If there is a charge at the protein surface, the protein prefers to interact
with water, rather than with other protein molecules. This charge makes it
more soluble.
e. Given the tripeptide aspartyl-alanyl-glycine (​asp-ala-gly)​:
e.1. Show the structure of the tripeptide in the presence of
e.1.1. Excess acid
e.1.2. Excess base
e.2. What is its isoelectric pH (ipH) -- acidic, basic, or neutral?

2. Biuret Test - Andrea

a. What is the principle involved in the biuret test?

- The Biuret test is based on the ability of Cu (II) ions to form a violet-coloured
chelate complex with peptide bonds in alkaline conditions. Lone electron pairs
from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion to form the
chelate complex.
b. What is the role of the reagents in the test? Illustrate with an equation and name the
compound responsible for the visible result.
- The blue reagent is made by combining sodium hydroxide and copper sulfate.
The role of these reagents turn the aqueous sample containing compounds with
peptide bonds form pale to violet depending on the concentration of the peptide
bonds on the amino acid.
c. WIll all the proteins give a positive biuret test? Why? WIll all peptides give a positive
biuret test? Explain.
 - Proteins will give a positive biuret test because all proteins contain peptide
bonds. There will be a violet colour to display the positivity, but the intensity
depends on the number of peptide bonds in the sample. However, not all
peptides will give a positive biuret test because at least 2 peptide bonds are
required for the formation of the chelate complex so dipeptides will give a
negative result.
d. Account for the difference in test results between egg white and aspartame (Equal).
Show the structure of aspartame to prove your point.
- The results for egg whites showed a violet colour while the results for aspartame
showed a pink color. This is because egg whites have proteins which have more
than 1 peptide bond and aspartame only has 1 peptide bond.

e. In the extraction of blood proteins, explain the function of the following reagents:
e.1. 23% Na2SO4 ​- The sodium sulfate is used to precipitate the globuline in the
blood.
e.2. Ether ​- The ethyl-ether is added to help separate the precipitate during
centrifugation.
f. What is the chemical composition of the precipitate at the interface after
centrifugation? Why should it not contaminate the tube marked A?
- Idk how to answer this (help)
g. Account for the difference in color intensity between samples TP and A.
- Idk this either :((((
h. What group in a peptide or a protein accounts for a positive Biuret test?
- CONH groups
i. Give the importance of the Biuret test in protein hydrolysis.
- Dipeptides do not give the biuret test, while all other polypeptides do so. Hence
biuret test is important to know whether hydrolysis of proteins is complete or not.
If the biuret test is negative, hydrolysis is complete, at least to the dipeptide
stage.

3. Ferric Chloride Test - leeza

https://www.quora.com/Whats-the-reaction-between-phenol-and-ferric-chloride
https://chemistry.stackexchange.com/questions/43187/what-is-the-product-of-the-chemic
al-reaction-between-phenol-and-ferric-chloride
a. Give the principle involved in the ferric chloride test. What is its purpose?
- Ferric chloride is used to determine the presence of phenols.
- Enols, hydroxamic acids, oximes and sulfinic acids give positive results as well.
 - However, the appearance of a color change on the addition of ferric chloride
depends to a great extent on such common factors as solvent, acidity, and
concentrations of reactants.
b. Identify the role of FeCl3 in this test.
- The ferric chloride forms a complex with the phenol, provided they are both very
dilute, and the complex, being a charge-transfer compound, happens to give a
very strong spectral signal in the visible, therefore it acts as a color test for
phenols.
c. What group/amino acid is responsible for a positive ferric chloride test?
- A positive ferric chloride test displays a violet precipitate
d. Illustrate the reaction involved in this test with an equation.
3ArOH+FeCl3⟶Fe(OAr)3+3HCl
The acid-base reaction and we get ferric phenolate salt as a precipitate. This acid-base
reaction results in a neutralization and a precipitate.
6ArOH+FeCl3⟶[Fe(OAr)6]3−+3H++3HCl
This is a ligand-exchange. A ligand is an ion or molecule attached to a metal atom by
coordinate bonding, or a molecule that binds to another (usually larger) molecule. A ligand
exchange is a type of chemical reaction in which a ligand in a compound is replaced by another.
Those three H+ ions are from the excess phenol. The ligand-exchange occurs only when
the phenol is in excess and more water molecules can be replaced, making the iron compound
ionic and soluble again.

4. Hopkins-Cole Test - leeza

a. Give the principle involved in the Hopkins-Cole test. What is its purpose?
- Also known as the glyoxylic acid reaction
- Detecting the presence of tryptophan in proteins
b. Give the composition of the Hopkins-Cole reagent and identify its role in the test.
- Magnesium glyoxylate made from a mixture of oxalic acid and magnesium

c. Show the equation involved in this test.

d. What compound is responsible for a positive result?

- The tryptophan that the Hopkins-Cole test determines is defined as an indole
nucleus and is known for creating the violet ring where the two layers meet.
 - Indole​ is an aromatic heterocyclic organic compound with formula C​8​H​7​N.
It has a bicyclic structure, consisting of a six-membered benzene ​ring
fused to a five-membered nitrogen-containing pyrrole ​ring​. ... The amino
acid tryptophan is an ​indole​ derivative and the precursor of the
neurotransmitter serotonin.
e. What group/amino acid present in a protein is identified by this test?
- Tryptophan

5. Ninhydrin Test - Andrea

a. Give the principle involved in the ninhydrin test. What is its purpose?
- Amines (including α-amino acids) react with ninhydrin to give a coloured product.
The purpose of the Ninhydrin test is to detect the presence of amino acids.
b. Identify the role of ninhydrin in this test.
- Ninhydrin is an oxidizing agent which leads to the oxidative deamination of
alpha-amino groups. Ninhydrin also reacts with primary amines however the
formation of carbon dioxide is quite diagnostic for amino acids.
c. Show the equation involved in this test.

-
d. What compound is responsible for a positive ninhydrin test?
- A positive ninhydrin test is either yellow or blue/violet. For a ninhydrin test to
appear yellow, there must be the presence of proline, which is a secondary
amine. For a ninhydrin test to appear blue/purple, there must be the presence of
α-amino acids
e. What group/amino acid in a protein is identified by this test?
- α-amino acids and proline
f. Did aspartame/Equal give a positive result? Why or why not?
 - Yes, it gave a blue/purple positive result. This was due to its composition
containing phenylalanine and aspartic acid
g. Will all amino acids give a positive ninhydrin test? Explain.
- No. The only amino acids which will have a positive result in the ninhydrin test
are proline and ​α-amino acids. β-amino acids and γ-amino acids will have
negative results in the ninhydrin tests.

6. Sakaguchi Test -- annika

a. What is the principle involved in this test? What is its purpose?
The Sakaguchi Test is used to determine the presence of In an alkaline solution, arginine reacts
with a-naphthol and sodium hypobromite /chlorite as an oxidizing agent to form red-colored
complexes as a positive result.
b. Why should NaOH be added first before the other reagents? Illustrate with an
equation the effect of adding NaOH to arginine.
c. What is the purpose of the other reagents?
In alkaline solution they give a red colour with the Sakaguchi reagent, which contains
α​-naphthol and sodium hypochlorite.

The quantity of sodium hydroxide employed may be varied considerably if a sufficient amount is
added to give the necessary alkalinity to the solution which is to be tested. An excess of
a-naphthol produces a yellow color, the intensity of which depends upon the amount used.
However the quantity of a-naphthol necessary to give complete color development with the
arginine concentrations employed, gives only the faintest trace of yellow and causes no
disturbance in color matching, even when very dilute solutions are compared.

The extremely sensitive color reaction given by certain guanidine derivatives with ar-naphthol
and sodium hypochlorite, which was observed by Sakaguchi (1925), has been extensively
employed as a qualitative test for arginine. The greatest obstacle in a quantitative application of
this reaction is its slow rate of color development with the probable destruction of some of the
guanidine group by the hypochlorite.
d. What group in arginine responds to this test? ​Guanidine group

7. Lead Acetate Test -- annika

a. Give the principle involved in this test. What is its purpose?
The lead acetate test is used to determine the presence of the sulfide group in
Cysteine. A positive test result will give a dark brown to black color after heating the solution
due to the lead ii sulfide compound
b. What is the role of NaOH in this test?
The sulphide group has to be free in order to be detected, so we must hydrolyse
the sample beforehand, using a hydrolysing agent, in this case we are using sodium
hydroxide (NaOH). When a sulphide group is detected, a brown colour is formed. When
lead acetate reacts with the free sulphide ions, the produce lead sulphide (PbS).
c. Illustrate with an equation the reaction between Pb(CH3COO)2 -- lead II acetate and
the product obtained after heating with NaOH.

d. What compound is responsible for the visible result?

Lead II Sulfide PbS
e. What group/amino acid is identified by this test?
The presence of the sulfide group in Cysteine.

The lead acetate test is used to determine the presence of sulfur in amino acids.
A. The precipitate is black. B. The product of this reaction is black lead (II) sulfide. It
forms upon the reaction between lead (II) acetate and the sulfur in the egg. C. dilute
sodium hydroxide in test tube: NaOH ---> Na+ + OH- addition of dry egg albumin: 2Na+
+ S ---> Na2S ---> 2Na+ + S2- addition of lead acetate: S2- + Pb2+ ---> PbS Because of
their peptide structure and the presence of different amino acid groups in their molecules
proteins react with a variety of agents to form colored products.
Principle: 
Sakaguchi test is Specific for free Arginine or proteins containing Arginine. 
Sakaguchi's test is positive for the amino acid containing the guanidine group in Arginine. 
Guanidine group present in the amino acid reacts with α-Naphthol and alkaline hypobromite 
to give red-coloured complex.
Sources:
http://people.uwplatt.edu/~sundin/351/351h-pro.htm​ -- biuret, xanthoproteic, HMI
http://fac.ksu.edu.sa/sites/default/files/color_test_for_specific_amino_acids.pdf
https://www.britannica.com/science/denaturation
http://chemistry.elmhurst.edu/vchembook/568denaturation.html
http://www.askmefast.com/What_happens_when_albumin_is_added_to_naoh_and_lead_acetat
e-qna696229.html
https://mtfiles.wordpress.com/biochemistry/color-reactions-of-proteins/
https://www.umb.edu.pl/photo/pliki/WL_jednostki/zaklad_biochemii_lekarskiej/pdf/biochemistry_
workbook.pdf
https://biology.stackexchange.com/questions/39931/why-is-70-ethanol-preferred-for-aseptic-tec
hniques
https://www.coursehero.com/file/15180986/Lead-Acetate-Test/
http://butane.chem.uiuc.edu/cyerkes/chem104A_S07/Lecture_Notes_104/lect27c.html
http://images.slideplayer.com/20/5938630/slides/slide_25.jpg
http://www.austincc.edu/mlt/chem/chemlab10proteins.pdf
https://www.cancer.org/cancer/cancer-causes/aspartame.html
http://community.preproom.org/index.php?threads/aspartame-and-the-biuret-test.394/
http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html
https://fulltimes.wordpress.com/protein/
http://intranet.tdmu.edu.ua/data/kafedra/internal/chemistry/lectures_stud/en/stomat/ptn/1/Bioorg
anical%20chemistry/05.%20Amino%20acids,%20peptides%20and%20proteins.htm
http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch27/ch27-3-3.html
http://www.jbc.org/content/25/2/319.full.pdf

Useful ones:
http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
http://people.uwplatt.edu/~sundin/351/351h-pro.htm
https://www.scribd.com/document/307023652/BioOrganic-Chemistry-Experiment-8-Characteriza
tion-of-Proteins-and-Amino-Acids
http://www.jbc.org/content/86/1/217.full.pdf
http://www.uobabylon.edu.iq/eprints/publication_5_3216_904.pdf