Granda, Arnold John – 3Chem-1

DATE: September 08, 2020

Experiment no. 2

Color Reactions of Intact Protein and Hydrolyzate

OBJECTIVE(S)

 To subject the intact proteins and hydrolyzates to various color reactions.

 To observe the color change that will occur.

MATERIALS AND INSTRUMENTS

A. Materials/Reagents

 Base Hydrolyzate

 Acid Hydrolyzate

 Intact Protein

 3 mL intact protein/hydrolyzate

 10 % 2.5 M NaOH

 0.01 M CUSO4

 10% NaOH

 0.02 % alpha-naphthol solution

 2% Hypobromite

 0.1% Ninhydrin Solution

 Concentrated HNO3
  Concentrated NaOH

 Concentrated H2SO4

 Hopkins Cole reagent

B. Instruments

 Erlenmeyer Flask

 Pipette

 Test tube

 Test tube rack

 Styrofoam

PROCEDURE

A. Biuret Test

3 mL Protein Suspension/Hydrolyzate

 + 1 mL 10% of 2.5 M NaOH solution

 + 1 mL of 0.01 M CUSO4

B. Sakaguchi Test

5mL Protein Suspension/Hydrolyzate

 + 1 mL of 10% 2.5 M NaOH solution

 + 1mL of 0.02 % alpha-naphthol solution
 + 1 mL of 2% Sodium Hypobromite.
C. Ninhydrin Test

1 mL Protein Suspension/Hydrolyzate

 + 0.5 mL of 0.1% Ninhydrin Solution

 Heat for 2-3 mins.

D. Xanthoproteic Test

1 mL Protein Suspension/Hydrolyzate

 + 3 drops of concentrated HNO3

 Heat solution for 1 min
 Cool in a flowing water
 + 0.5 mL of concentrated sodium hydroxide

E. Hopkins-Cole Test
2 Drops Protein
Suspension/Hydrolyzate

 + 1 mL Hopkin’s Cole reagent

 + 1 mL concentrated H2SO4 to an inclined tube (Slowly)
RESULTS

A. Biuret Test

o The color of the intact protein upon the addition of 1 mL 10% of 2.5 M

NaOH solution and 1mL 0.01 M CUSO4 from white, it became violet and

translucent. While the acid and base hydrolyzate turned light yellow and

pinkish violet respectively. The three solutions became translucent.

B. Sakaguchi Test

o The color of the intact protein upon the addition of 1 mL 10% of 2.5 M

NaOH solution, 1mL of 0.02 % alpha-naphthol solution and 1 mL of 2%

Hypobromite from white, it became red. While the acid and base

hydrolyzate turned yellowish brown and beige respectively. The three

solutions became translucent.

C. Ninhydrin Test

o The color of the intact protein upon the addition of 0.5 mL of 0.1%

Ninhydrin Solution from white, it became cloudy white translucent. While

the acid and base hydrolyzate were turned in to a dark red color. The

reacted acid hydrolyzate turned into dark brown color, while the unreacted

acid hydrolyzate turned into a gold color. The reacted base hydrolyzate

became dark red color, while the unreacted base hydrolyzate turned in to

an orange color.
D. Xanthoproteic Test

o The color of the intact protein upon the addition of 3 drops of concentrated

HNO3 mL and 0.5 mL of concentrated sodium hydroxide from white, it

turned yellow. While the acid and base hydrolyzate turned brown and

orange respectively. The three solutions became translucent.

E. Hopkins-Cole Test

o The color of the intact protein upon the addition of 1 mL Hopkin’s Cole

reagent and 1 mL concentrated H2SO4 from white, it turned into colorless

liquid with a tinge of purple color at the upper portion of the solution.

While the acid and base hydrolyzate turned light yellow and light orange

respectively. The three solutions became translucent.

EXPERIMENT 2 – COLOR REACTIONS OF INTACT PROTEIN AND

HYDROLYZATE

1. Give the principle behind each chemical test done

A. Biuret Test

o The principle behind Biuret test is the determination of the peptide bonds
in a protein. It is based on the ability of Cu (II) ions to form violet colored
chelate complex with the peptide bonds in alkaline conditions. Absorption
of light at around 540 nm happens in the chelate complex, this is the
reason it appears violet which means that proteins are present. The higher
the concentration of peptide bonds = higher or greater the color intensity.
B. Sakaguchi Test

o The principle behind Sakaguchi test is that it gives a color red product
because the protein/arginine reacts with α-naphthol in the presence of an
oxidizing agent such as sodium hypobromite to give a red coloured
product.
C. Ninhydrin Test
o The principle behind Ninhydrin test is the reaction between an amino
group with a free amino acid and ninhydrin to give a purple complex
product. The presence of a strong oxidizing agent such as ninhydrin which
makes the amino acid undergo oxidative deamination, thus freeing
ammonia, C02, aldehyde, and the reduced form of ninhydrin.
D. Xanthoproteic Test

o The principle behind Xanthoproteic test is that it uses a nitration reaction

in order to determine the protein content in a solution. It produces a yellow
colored solution upon heating with conc. HNO3.

E. Hopkins-Cole Test
 o The principle behind the Hopkins-Cole test is that a reaction between the
indole group of tryptophan and gyoxilic acid with the presence of
concentrated H2SO4 produces a purple colored solution.

2. Compare the results with the acid and base hydrolyzates. Explain the differences.

A. Biuret Test

o The acid hydrolysate had a light yellow color while the base hydrolysate

had a pinkish color.

B. Sakaguchi Test

o The acid hydrolysate had a yellowish brown color while the base

hydrolysate had a beige color.

C. Ninhydrin Test

o The acid and base were both dark red in color. The reacted acid

hydrolyzate turned into dark brown color, while the unreacted acid

hydrolyzate turned into a gold color. The reacted base hydrolyzate became

dark red color, while the unreacted base hydrolyzate turned in to an orange

color.

D. Xanthoproteic Test

o The acid hydrolysate had a brown color while the base hydrolyzate had an

orange color.

E. Hopkins-Cole Test

o The acid hydrolysate had a light yellow color while the base hydrolyzate

had a light orange color.