Notre Dame of Dadiangas University

College of Health Sciences

Bachelor of Science in Medical Technology
NAME: ARQUISOLA, BARANDA, PAPASIN, YR &SEC: BSMT – 2A
PURIFICACION, VENCER
PROFESSOR: MS. ELIZABETH C. BILLS DATE: SEPTEMBER 22, 2020

STUDY GUIDE NO. 2

PROTEINS (PART 1)
A. PROTEIN DENATURATION – ARQUISOLA AND PURIFICACION
1. Define denaturation. – PURIFICACION
- Denaturation is defined as the alteration of a protein shape through some form of
external or visible stress (e.g. applying heat, acid or alkali), in such a way that it
will no longer function properly.

2. What physical and chemical agents are capable of denaturing proteins? Give the type
of bonds or attractive interruptions disrupted by these denaturing agents. -
PURIFICACION
- In physical agents, these include: heat, surface action, ultraviolet light, ultrasound,
high pressure etc. While in chemical agents, Acids, alkalis, heavy metal salts, urea,
ethanol, guanidine detergents etc.

- The type of bonds that are disrupted by these denaturing agents are the bonds in
the secondary structure which is hydrogen bonding to amides. Secondly, are the
bonds in tertiary structure. The bonds that are present in tertiary structure
hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic
interaction

3. What concentration of alcohol is most effective as a disinfectant? Why?-ARQUISOLA

- 70% isopropyl alcohol is the most effective concentration of a disinfectant

because this solution kills microorganisms by dissolving plasma membrane of the cell wall.
Water is essential to denature the proteins of the cell membrane and act as a catalyst,
70% isopropyl alcohol solution penetrates in the cell wall and coagulates the all protein of
the cell wall and microorganism dies

4. Explain how protein denaturation using heat, alcohol, and heavy metal ions is used in
the medical field.-ARQUISOLA

- Protein denaturation using heat is used to sterilize the medical supplies and tools to
eliminate microorganisms and bacteria that will cause infection due to blood
contamination. Protein denaturation using alcohol is used in the medical field to
prevent the contamination of microorganisms by killing them through penetrating the
cell wall and coagulates all gram-negative bacteria.

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.
 - Also, Protein Denaturation using heavy metal ions is used in the medical field as
disinfectant properties in an external application, AgNO3 is used to prevent gonorrhea
infections in the eyes of the newborn infants. Silver nitrate is also used to treat nose
and throat infections.

5. What amino acids in a protein are reactive with heavy metal ions? Explain and illustrate
with an equation. -PURIFICACION

- Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals
with high atomic weights. 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.

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.
 - Heavy metals may also disrupt disulfide bonds because of their high affinity and
attraction for sulfur and will also lead to the denaturation of proteins. If oxidizing
agents cause the formation of a disulfide bond, then reducing agents, of course,
act on any disulfide bonds to split it apart.

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.
 B. COLOR REACTIONS
1. XANTHOPROTEIC REACTION - PAPASIN
1. Give the principle involved in this test.
- It is used to test Amino acid, mainly the Aromatic nucleus. The Aromatic
nucleus/ Benzene ring undergoes nitration to give off yellow substance
as their end product. Adding alkali on the nitro derivative salts, it
changes from yellow to orange.

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

- Aromatic Benzene / Aromatic Nucleus

3. Name the amino acid that will be positive with this test.
- Tyrosine, Tryptophan, and Phenylalanine. But, Phenylalanine may be
negative or weakly positive in the test.

4. What is the isoelectric point? Explain how pH changes in a protein solution

affect its solubility.

- Isoelectric point is when a specific pH (negative and positive charges)

will be balance and there will be a net charge of 0. pH affects the
solubility by changing the charge state of the solute. If the pH has no
net charge, then the solute will have minimal solubility and precipitates
in the solution.

2. BIURET TEST -VENCER

1. What is the principle involved in the biuret test?
- A chemical test used to determine the presence of proteins, specifically
peptide bonds within the given sample.

2. What is the role of the reagents in the test? Name the compound responsible
for the visible result.
- Potassium hydroxide and Copper sulfate

The copper ions found within the reagent that contains a charge of +2, are
reduced to a charge of +1 due to the presence of peptide bonds. This later causes
the color change. Based on absorption spectroscopy, this identifies the
electromagnetic frequencies that the sample will absorb, which allows testers to
determine the concentration of the protein within a sample.

3. Will all proteins give a positive biuret test? Why? Will all peptides give a positive
biuret test? Explain.

- Biuret test is used for the presence proteins. Peptide bonds (C-N bonds)
in proteins complex with Cu2+ causes the reagent and produce a violet color. A
Cu2+ must be in complex with four to six peptide bonds in order to positively react
for the test.

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.
 4. What group in a peptide or a protein accounts for a positive Biuret test?

- This test is given by compounds containing two or more peptide bond

(CO-NH group). Since all proteins and peptides possess at least two peptide
linkage, tripeptide gives the positive result for the biuret test.

5. Give the importance of biuret test in protein hydrolysis.

-The importance of studying hydrolysis of proteins by the use of Biuret test

is that this is where we can determine the concentration of protein not based on
presence of different kinds of amino acids alone, but also based from the
qualitative assessment for the amount or the functional activity of a target entity.

3. FERRIC CHLORIDE TEST -BARANDA

1. Give the principle in the ferric chloride test. What is its purpose?

- The sample is dissolved in water, or a mixture of water and ethanol, and a few
drops of dilute ferric chloride (FeCl3) solution is added. The formation of a red,
blue, green, or purple coloration indicates the presence of phenols. is used to
determine the presence of phenols.

2. Identify the role of ferric chloride in this test.

- It reacts with the presence of phenols and it is the one the changes the color of
the sample when phenol compound is known.

3. What group/amino acid is responsible for a positive FeCl2 test?

- Fe3+

4. Illustrate the reaction involved in this test with an equation.

2Fe + 3Cl2 → 2FeCl3

(chemical equation)

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.
 1. By dissolving iron ore in HCl (hydrochloric acid)

Fe3O4 + 8HCl → FeCl2 + 2FeCl3 + 4H2O

2. By oxidizing iron (II) chloride with chlorine (Cl)

2FeCl2 + Cl2 → 2FeCl3

3. By oxidizing iron (II) chloride with oxygen

4FeCl2 + O2 + 4HCl → 4FeCl3 + 2H2O

REFERENCES:
A.PROTEIN DENATURATION
https://www.sciencedaily.com/terms/denaturation_(biochemistry).htm
https://www.biologydiscussion.com/proteins/denaturation-of-proteins-with-denaturing-
agents/41902
http://chemistry.elmhurst.edu/vchembook/568denaturation.html
http://chemistry.elmhurst.edu/vchembook/568denaturation.html
https://www.pharmaguideline.com/2013/11/why-70-isopropyl-alcohol-is-used-as-
disinfectant.html

http://chemistry.elmhurst.edu/vchembook/568denaturation.html

B. XANTHOPROTEIC REACTION
https://courses.lumenlearning.com/introchem/chapter/the-effect-of-ph-on-solubility/

http://www.chemistryexplained.com/Pr-Ro/Protein-Solubility.html

BIURET TEST

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.
Eric Bank, M. (2017, November 21). What Does a Biuret Test Mean in Biology? Retrieved
September 21, 2020, from https://education.seattlepi.com/biuret-test-mean-biology-4659.html

Karki, G. (2020, January 20). Biuret test-Principle, Requirements, Reagents, Preparation,

Procedure and Result. Retrieved September 22, 2020, from
https://www.onlinebiologynotes.com/biuret-test-principle-requirements-reagents-preparation-
procedure-and-result/

Libretexts. (2020, July 14). 2.9: Proteins. Retrieved September 22, 2020, from
https://bio.libretexts.org/Bookshelves/Biotechnology/Bio-
OER_(CUNY)/02:_Chemistry/2.09:_Proteins

Munez, C. (n.d.). 2A Final - Group 4. Retrieved September 22, 2020, from

https://www.scribd.com/document/93305640/2A-Final-Group-4

FERRIC CHLORIDE TEST

https://en.wikipedia.org/wiki/Ferric_chloride_test#:~:text=The%20sample%20is%20dissolved%2
0in,indicates%20the%20presence%20of%20phenols.
https://www.genscript.com/molecular-biology-glossary/1080/ferric-chloride-
test#:~:text=The%20chelation%20of%20Fe3%2B%20by,levels%20of%20phenylalanine%20in%
20phenylketonuria.
https://byjus.com/chemistry/ferric-
chloride/#:~:text=Ferric%20Chloride%20is%20a%20dark,Ferric%20Chloride%20is%20FeCl3.

Reference: Piedad C. Pena (et al.) Biochemistry laboratory manual – 1st ed. – Quezon City: C&E
Publishing, Inc., 2015.