St.

Paul College of Ilocos Sur

(Member, St. Paul University System)
St. Paul Avenue 2727, Bantay, Ilocos Sur

NSC 101- BIOCHEMISTRY

LABORATORY NOTES

Prepared by: KATYA AMELIA A. VALIDO, LPT

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 TESTS FOR PROTEINS
➢ Proteins are complex biomolecules composed of alpha amino acids joined by the peptide
linkages/bond.

➢ A peptide bond (an amide bond) formed by the loss of a molecule of H2O from the -COOH
group of one of the amino acids and the -NH2 group of an adjacent amino acid.

➢ The building blocks that make up proteins are called amino acids.

Prepared by: KATYA AMELIA A. VALIDO, LPT

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➢ Essential amino acids cannot be made by the body. As a result, they must come from
food. Essential amino acids like; histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine.
➢ Non-essential amino acid means that our body can produce the amino acids, even, if
we do not get it from the food we eat.
➢ Conditional essential amino acids only considered functional in times, of illness, stress,
and pregnancy.

➢ R is any aliphatic or aromatic radical. Changing the R changes the identity of the amino
acid. The alpha carbon has a (+) charge, and the (H +) attached to its slightly acidic.

Prepared by: KATYA AMELIA A. VALIDO, LPT

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➢ PROPERTIES OF AMINO ACIDS
1. They are soluble in water, insoluble in organic solvents (like benzene, ether, etc.).
2. They have large dipole moments and dielectric constants.
3. Amino acids are polar.
4. Polar forms of an amino acid are called zwitterions.

5. Amino acids may be acidic (-COOH), basic (NH2), or neutral.

➢ CHEMICAL REACTIONS OF AMINO ACIDS

- Due to the presence of two functional groups, the -COOH group and the -NH2
group, amino acids are amphiprotic.

➢ COLOR REACTIONS OF PROTEIN AND AMINO ACID

1. Xanthoproteic Test
Purpose:
This test is used for aromatic amino acids, which give positive result from other
amino acids.
Principle:
- To detect amino acids containing an aromatic nucleus (tyrosine, tryptophan,
and phenylalanine) in a protein solution which gives yellow color nitro
derivatives on heating with conc. HNO3.
- The aromatic benzene rings undergo nitration to give yellow colored product.
- Phenylalanine gives negative or weakly positive reaction though this amino
acid contains aromatic nucleus because it is difficult to nitrate under normal
condition.
- On adding alkali to these nitro derivative results the color change for yellow to
orange.

Prepared by: KATYA AMELIA A. VALIDO, LPT

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 Result:
(+) Yellow colored product (proteins with tryptophan, tyrosine, and phenylalanine)
(-) Absence of yellow or orange colored product

2. Biuret Test/Piotrowski’s Test

Purpose:
- It is a general test that can be used to check for the presence of peptide bonds
in a given analyte.
Principle:
- When biuret is treated with copper sulfate (CuSO4) in the presence of alkaline,
a purple-colored material is generated.

Result:
(+) Formation of purple color (analyte contains protein)
(-) Formation of light blue color (the protein concentration is low and considered
a negative result)

Prepared by: KATYA AMELIA A. VALIDO, LPT

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Buiret Test: FAQs
1. Why do we need the Biuret test?

2. Why is Biuret purple in color?

3. Hopkin’s -Cole Test/ Glyoxylic Acid Test

Purpose:
- It is a specific test used for the detection of indole ring.
- To detect the presence of tryptophan-containing proteins.
Principle:
- The layering of conc. H2SO4 over a mixture of tryptophan-containing proteins
with the Hopkin’s Cole reagent results in the formation of a violet ring at the
interface.

Prepared by: KATYA AMELIA A. VALIDO, LPT

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 Result:
(+) Formation of purple-colored ring at the junction of two layers; indicates the
presence of amino acid tryptophan.
(-) Absence of a purple-colored ring; indicates the absence of tryptophan.

4. Ninhydrin Test

Purpose:
- It is a chemical test which is used to check whether a given analyte contains
amine (-NH2)
Principle:
- The amino acid belonging to a free amino acid undergoes a chemical reaction
with ninhydrin.
- When exposed to ninhydrin, the amino acid undergoes oxidative deamination,
resulting in the liberation of CO2, NH3, and an aldehyde along with
hydrindantin.
- Now, the NH3 goes on to react with another ninhydrin molecule to form
diketohydrin (Ruhemann’s complex).
- This complex is responsible for the deep blue color, yellow color (proline).

Result:
(+) Deep blue color (contains amino acid)
Yellow (contains proline)
(-) No color change (absence of amino acid)

Prepared by: KATYA AMELIA A. VALIDO, LPT

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Ninhydrin Test: FAQs
1. Why is Ninhydrin used by fingerprints?

2. What is the purpose of ninhydrin reagent?

5. Sakaguchi Test

Purpose:
- It is a biochemical test consisting of colorimetric reaction for the detection and
quantification of guanidinium groups used a qualitative test for Arginine (Arg).
Principle:
- Sakaguchi test is based on the principle of reaction between 1-naphthol and
the guanidinium groups in arginine, in the presence of an oxidizing agent.
- The reaction results in the formation of a red-colored complex due to the
formation of an indole-like structure.
- L-arginine + alpha-naphthol + NaBrO yields to red compound

Result:
(+) Formation of red color. This indicates the presence of an arginine (Arg) or
guanidinium compound.
(-) Demonstrates the absence of red color. This indicates an absence of
arginine or a guanidinium compound.

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6. Nitroprusside Test

Purpose:
- The test is a specific test for cysteine in a protein solution.
- The test can also be used for the differentiation between cysteine and cystine.
Principle:
- Nitroprusside test is based on the detection of 5-sulfur atoms in a solution as
result of the degradation of the -SH group.
- The nitroprusside when combined with a sulfur atom forms red-colored
complex.

Result:
(+) A positive result in the nitroprusside test is indicated by the appearance of
red-colored complex. The color formation confirms the presence of cysteine.
(-) A negative result in the nitroprusside test is indicated by the absence of a
red-colored solution. This indicates the absence of cysteine.

Prepared by: KATYA AMELIA A. VALIDO, LPT

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➢ USES OF NITROPRUSSIDE TEST
- This test can also be used in the diagnosis of cystinuria as the presence of
cysteine in urine is a pathological feature of the disease.
- The nitroprusside test also detects compounds like ketoacids and ketones in
blood and urine that helps in the determination of the degree of ketonuria and
ketonemia.

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 PROTEIN DENATURATION
➢ Denaturation
- Refers to any disruption in the secondary, tertiary, and quaternary levels of
protein structure.
- It does not cleave the peptide bonds; therefore, the primary level of structure
is not altered.
- Physical signs of denaturation in a protein are precipitation and coagulation.

➢ COMMON AGENTS OF DENATURATION

1. Heat and UV radiation
- Thermal agitation due to additional kinetic energy in the molecules makes it
vibrate more.
- This breaks the H+ bonds, causing the folded structure of proteins to uncoil or
unwind into random loops.

➢ Why do we cook our food?

- Sterilization procedures make use of this to kill bacteria, and the cooking of
food to make the food more digestible.
- For example: egg albumin protein

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2. Alcohol disrupts H+ Bond
- Hydrogen bonding occurs between amide groups in the secondary protein
structure.
- A 70% alcohol solution is used as a disinfectant on the skin. This concentration
of alcohol is able to penetrate the bacterial cell wall and denature the proteins
and enzymes inside of the cell.

3. Heavy Metals
- Heavy metal salts usually contain Hg, Pb, Ag. The reaction of a heavy metal
salt with a protein usually leads to an insoluble metal protein salt.
- Applications include the use of AgNo3 to prevent gonorrheal infection in the
eyes of the newborn, use of HgCl2 for skin infections, and the use of egg
albumin as antidote for heavy metal poisoning.

4. Acids and Bases

- Acid and bases can significantly change the environmental pH of proteins,
leading to denaturation
- If the protein is subject to change in pH, the internal interactions between the
proteins amino acids can be altered, which in turn may alter the shape of the
protein.

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References:
Pena, P.C., Garcia, M.R.V., Saludares, M. D., Navidad, F. C., Villaflores, O. B., Dicioco, C. C.,
(2015). Biochemistry Laboratory Manual. C&E Publishing Inc.
Stoker, H. S. (2017). Biochemistry Third Edition. C&E Publishing Inc.

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