casein

Two or more amino acids bonded together by peptide bonds (covalent bonds)

Production of enzymes, hormones Protein catabolism- breakdown of proteins

Can be classified based on : Levels of protein structure (Primary,

Secondary, Tertiary, Quaternary) Structure (Fibrous, Globular) Composition (Simple, Conjugated)

Building blocks of proteins 20 are usually present in proteins Molecules that contain an amino group,

carboxyl group, Hydrogen atom and R group attached to the a- carbon

R groups are based on two factors: Polarity, Acidic or Basic.

Main protein in milk 80% protein present in milk

Uses strong acids (H2SO4, HCl) Split the peptide bonds of the proteins

Uses strong bases (NaOH, Ba(OH)2) Breaking of peptide bonds of the protein

Methods Determination of the presence of amino

acids and proteins

To identify the components present in the protein by means of performing chemical tests.

Casein Biuret Test

-Casein ANY PROTEIN

NET CHARGE pI = 0

SALT

Sakaguchi Test

Ninhydrin Test

ARGININE

AMINO GROUPS NH

Xanthoproteic test

Activated Benzene Rings Deactivated

Hopkins-Cole Test

Color Reactions

Mortar and pestle Spot plate Acid/base hydrolyzate (neutralized) 2.5 M NaOH 0.01 M CuSO4 solution 10% NaOH 0.02% naphthol solution NaOBr (freshly-prepared)

0.1% ninhydrin solution

Conc. HNO3 Conc. NaOH Hopkins-Cole

reagent Conc. H2SO4

Preparation of Protein Suspension

Dried casein

Cut into small pieces

Place in mortar

Add 10 mL d. H2O

Grind

Protein Suspension

Protein Suspension/Hydrolyzate (3 drops)

B.1 Biuret test

Add 1 drop of 2.5 M NaOH

Mix Add at least 1 drop of 0.01 M CuSO4 solution

Mix

(+) Pink to violet to blue solution

Color Reactions


Add 1 drop 10% NaOH

Add 1 drop of 0.02% naphthol solution

Mix

Leave for 3 mins.

Add 1 drop of freshly-prepared 2% NaOBr

(+) Red to red-orange solution

Color Reactions

B.2 Sakaguchi test

Color Reactions

B.3 Ninhydrin test

Protein Suspension (10 drops)/Hydrolyzate (1 mL)

In protein suspension: add 1 mL water

Add 0.5 mL of 1% ninhydrin solution

Mix

Heat in boiling water bath for 2-3 mins.

(+) Blue to blue violet solution

Color Reactions

B.4 Xanthoproteic test

Protein Suspension (10 drops)/Hydrolyzate (1 mL) In protein suspension: add 1 mL of water

Slowly add 3 drops of conc. HNO3

Mix

Heat in boiling water bath for 1 min.

Cool solution with flowing water

Slowly add conc. NaOH drop by drop until solution is alkaline

(+) Yellow-orange precipitate

Color Reactions

B.5 Hopkins-Cole test


Add 2 mL of Hopkins-Cole reagent

Mix

Slowly add 2 mL conc. H2SO4

(+) Pink to violet ring at interphase

Results and Discussion of protein hydrolyzate

Tryptophan completely destroyed and turned into humin

After autoclave: brown-black solution with black precipitate (Tryptophan )

Autoclaving destroys arginine, cysteine, threonine, and serine

After autoclave: yellow liquid with white mass

Principle: complexation of Cu2+ atoms with amide N atoms

The biuret reagent (copper sulfate in strong base)reacts with peptide bonds in proteins to form a blue to violet complex known as the biuret complex

Biuret complex

B A S E

Group 2 Violet solution

Group 4 Blue solution

Group 6 Violet solution with white ppt

Group 8 Clear violet solution with ppt

Group 10 Purple solution

A C I D

Group 1 Clear light blue-violet liquid

Group 3 Clear blue solution with some ppt

Group 5 Blue-violet solution

Group 7 Light blue solution


Group 11 Clear blue solution

Acid (-) Base (-) No reaction for both, peptide bonds were broken during hydrolysis

Mechanism

Red or red-orange

-napthol Arginine

Principle: Base-catalyzed condensation of -napthol with the guanidino group of arginine

Base catalyzed because arginine only reacts at alkaline environment

A C I D

Group 1 light yellow liquid

Group 3 Clear yellow solution

Group 5 Yellow-orange solution

Group 7 Yellow-brown solution

Group 9 Yellow solution

Group 11 Clear light yellow solution

B A S E



Group 6 Clear light yellow solution with white precipitate

Group 8 Clear yellow solution

Group 10 Clear solution

Acid (+) Base (-) -napthol reacted with arginine

No reaction, arginine destroyed during hydrolysis

Ruhemanns purple

Principle: Oxidative deamination and decarbolxylation; reduction of ninhydrin

Ninhydrin reacts with -amino acids (-NH2) in proteins, producing Ruhemanns purple (purple complex), except in Proline and hydroxyproine which produces a yellow color

A C I D

Group 1 Clear orange liquid

Group 3 Red-orange opaque solution

Group 5 Dark orange solution

Group 7 Clear brown solution with brown ppt


Group 11 Clear reddish-violet solution

B A S E

Group 2 Violet solution with ppt

Group 4 Red solution

Group 6 Cloudy Violet solution

Group 8 Brown solution

Group 10 Cloudy solution

Acid Base

Acid (+) Base (+) Free -amino group reacts with ninhydrin

Principle: nitration of aromatic ring via electrophilic aromatic substitution

Tyrosine and tryptophan reacts with conc. HNO3 at high temperature forming the nitro compound which is yellow in color

Xanthoproteic Acid

Upon the addition of a base (NaOH), the yellow solution turned orange

Base was used because the aromatic group ionizes at alkaline environment

A C I D

Group 1 Clear yellow liquid; clear orange liquid

Group 3 Orange solution with suspensions

Group 5 Clear Orange solution

Group 7 Yellow-orange solution with ppt

Group 9 Yellow-orange solution

Group 11 Clear Yellow-orange solution

B A S E


Group 4 Orange solution blue litmus paper

Group 6 Milky solution

Group 8 Clear Yellow solution

Group 10 Golden yellow solution

Acid (+) Base (+) Nitration of tyrosine Nitration of tryptophan

Principle: reduction of oxalic acid to glyoxilic acid and acid-catalyzed condensation of tryptophan with glyoxilic acid

Violet ring: tryptophan and glyoxilic acid

A C I D

Group 1 Cloudy upper layer, clear lower layer

Group 3 Opaque colorless soln at the bottom, clear colorless on top

Group 5 Clear colorless solution


Group 9 Clear colorless solution

Group 11 Cloudy white solution

B A S E


Group 4 White upper layer, clear yellow lower layer

Group 6 No violet ring formed



Acid (-) Base (+) No violet interphase Formation of violet interpahse

indicating presence of Try

Protein suspension - GROUPS 1,3,5,7,9,11 | RESULTS

Type of test

1 3 5 7 9 11

Biuret

Clear blue violet solution

Clear violet solution

Light violet solution

Violet solution Violet solution Violet solution

Sakaguchi Light yellow liquid solution

Clear light yellow liquid solution

Yellow liquid solution

Very light yellow liquid solution

Light yellow liquid solution


Ninhydrin

Clear violet liquid solution

Violet solution with precipitate

Clear colorless solution with white precipitate

Clear solution with violet precipitate

Light violet precipitate in cloudy solution

White solution with white precipitate

Xanthoproteic

Clear yellow solution

Clear orange solution

Clear yellow orange solution w/ yellow precipitate

Yellow precipitate in clear yellow orange solution

Light yellow precipitate in cloudy solution

Yellow orange solution

Hopkins-Cole

Violet ring at interphase

Violet ring w/ precipitate at top

Transparent violet solution w/ violet ring

Cloudy upper layer, violet ring at interphase,

Clear solution with violet ring at interphase

Violet solution with violet ring

Protein suspension - GROUPS 2,4,6,8,10 | RESULTS

Type of test

2 4 6 8 10

Biuret

Violet solution Violet solution Violet solution Violet solution Violet solution

Sakaguchi Yellow liquid solution


Clear colorless solution

Turbid solution Clear liquid solution

Ninhydrin

Violet solution with precipitate

Violet liquid solution

Cloudy violet solution

Light violet solution with violet precipitate

Cloudy solution

Xanthoproteic

Yellow precipitate

Upper: white solution Lower: yellow orange precipitate

Clear yellow orange solution w/ white precipitate

Clear orange solution

Yellow precipitate

Hopkins-Cole

Cloudy upper layer, clear bottom layer

White translucent upper layer, clear bottom layer

Violet ring at interphase

Cloudy upper layer, violet ring at interphase, clear bottom solution

Violet ring

The complex of Cu2+ ions and N atoms makes the color of the solution change from blue to a violet color (Chelate complex)

The intensity of the color is directly proportional to protein concentration

(+) result is purple or violet Positive in: Intact Casein

Negative in: Base and Acid Hydrolyzate

Guanidine group present in the amino acid reacts with -Naphthol and alkaline hypobromite to give red-coloured complex.

(+) result is red to red-orange color Positive in: Intact Casein and Acid

Hydrolyzate Negative in: Base Hydrolyzate

Positive in: Intact Casein and Acid Hydrolyzate

Negative in: Base Hydrolyzate

(+) result is a blue-violet to purple solution (Ruhemanns purple)

While most ninhydrin tests result in a purple color, the proline reaction is more yellow due to substitution of the alpha amino group that ninhydrin reacts with. This has to do with carbon rings.

Positive in: Acid and Base hydrolyzate Negative in: Intact Casein

(+) yellow to orange precipitate (+) result in this test produces YELLOW color salts formed from the derivatives produces

ORANGE color (+) Intact casein, base hydrolyzate and acid

hydrolyzate

Once the tryptophan is free, the indole ring reacts with glyoxylic acid in the presence of a strong acid to form violet interphase

Positive result in this test yields purple ring in the boundaries of the solution

(+)Intact casein and base hydrolyzate

The presence of amino acids in casein was determined using color reaction tests.

Intact casein contains: peptide bonds, aromatic amino acids, tryptophan, arginine

Acid hydrolyzate contains: -amino group, aromatic amino acids, arginine

Base hydrolyzate contains: -amino group, aromatic amino acids, tryptophan

Casein was not very dry (preparation of protein suspension).

Addition of wrong reagents. Excessive or lacking amount of

suspension/hydrolyzate/reagent was added.

casein

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