PROTEIN

 Intact
protein- have a fixed conformation with no
peptide bond breakage.

 Hydrolyzate
protein- have disrupted structural
conformation due to hydrolysis

 Method: Hydrolysis and Analysis of color reactions

 Hydrolysis
-is a reaction involving the breaking of a bond
in a molecule using water.
 Color Reactions
- tests that enable to identify the different
properties of the sample protein using different
reagents
 Interphase
- is the intermediate phase between two
materials in equilibrium, which will join to form the
composite.
 To characterize the casein and acid/base
hydrolyzate using various chemical tests
 Casein
Figure 1. Schematic diagram of
Methodology - Cut dried casein & put in mortar
- Add 10 mL distilled water and grind
Protein Suspension

Biuret Test Sakaguchi Ninhydrin Xanthoproteic Hopkin’s-Cole

Test Test Test Test
- 1 drop 2.5 M
NaOH to 3 drops - 1 drop 10% -10 drops protein -10 drops protein -1 mL Hopkin’s Cole
protein NaOH + 1 drop suspension + 1 mL suspension + 1 mL water reagent to 2 drops
suspension/ 0.02% naphthol water -acid base hydrolyzate protein
hydrolyzate solution to 5 drops -Acid/ Base use 1 mL w/o dilution suspension/hydro-
-add drop 0.01M protein hydrolyzate use 1 mL with water lyzate
CuSO4 suspension/hydro- w/o dilution with -add 3 drops conc. -Incline test tube 45
-Mix lyzate water HNO3 degrees & slowly
-Mix -add 0.5 mL 0.1% Heat for 1 min (boiling add 2mL conc.
-3 min: 1 drop Ninhydrin solution water bath) H2SO4 (down the
freshy prepared -Heat for 2-3 min -Cool & slowly add conc. side)
2% NaOBr (boiling water bath) NaOH drop by drop until -two layers are
alkaline formed (interphase)

Result
 Sakaguchi Ninhydrin
Biuret Test
Test Test
- 1 drop 2.5 M - 1 drop 10% NaOH + 1 -10drops protein
NaOH to 3 drops drop 0.02% naphthol suspension + 1 mL water
protein solution to 5 drops -Acid/ Base hydrolyzate
suspension/ protein use 1 mL w/o dilution with
hydrolyzate suspension/hydro-lyzate water
-add drop 0.01M -Mix -add 0.5 mL 0.1%
CuSO4 -3 min: 1 drop freshy Ninhydrin solution
-Mix prepared 2% NaOBr -Heat for 2-3 min (boiling
water bath)
Xanthoproteic Hopkin’s-Cole
Test Test
-10drops protein -1 mL Hopkin’s Cole reagent
suspension + 1 mL water to 2 drops protein
-acid base hydrolyzate suspension/hydro-lyzate
use 1 mL w/o dilution with -Incline test tube 45 degrees
water & slowly add 2mL conc.
-add 3 drops conc. HNO3 H2SO4 (down the side)
Heat for 1 min (boiling -two layers are formed
water bath) (interphase)
-Cool & slowly add conc.
NaOH drop by drop until
alkaline
Table 1. Result of Biuret Test (groups 1-11)

BIURET TEST
GROUP ACID/BASE HYDROLYZATE
INTACT CASEIN
#
1 light Purple solution Yellow to blue color

2 clear Violet solution clear Blue Violet solution

3 clear Purple liquid Green solution w/ Brownish ppt

4 light Violet solution Blue solution

5 Violet solution Green solution

6 clear Purple solution turbid blue solution with precipitate

7 Violet Blue Green clear solution

8 Milky/White color > clear Violet solution clear Blue solution

9 clear Violet solution clear olive Green solution

10 Purple solution clear Bluish solution

11 White liquid > Purple Yellowish liquid > Blue-green

Table 2. Result of Sakaguchi Test (groups 1-11)

SAKAGUCHI TEST
GROUP ACID/BASE HYDROLYZATE
INTACT CASEIN
#
1 Yellow-orange w/ a red dot Orange liquid

2 clear Red solution clear Red Orange solution

3 Red Orange solution w/ Reddish ppt Yellow solution

4 Light Red solution light Yellow Solution

5 Red solution Yellow solution

6 clear red solution turbid yellow solution with precipitate

7 clear solution > Red Orange no color change

8 Milky/white color > Red no color change

9 clear Red Orange solution no color change

10 clear Red Orange solution clear Yellowish solution

11 Red solution no color change

Table 3. Result of Ninhydrin Test (groups 1-11)
NINHYDRIN TEST
GROUP ACID/BASE HYDROLYZATE
INTACT CASEIN
#
1 Light violet soln. w/ cloudy upper layer Dark violet to brown
2 clear Blue Violet ppt clear Brown solution
3 cloudy solution w/ Purple ppt rusting in the Violet > Brown solution
bottom
4 transparent solution w/ White ppt at the clear brown solution
bottom After heating: Violet solution
5 light Purple solution w/ White precipitate clear dark Red solution
6 turbid Blue Violet solution clear dark Red solution
7 no color change; white ppt interphase color:Brownish Red
8 presence of ppt, turbid liquid, interphase interphase; Upper: Violet, Middle:
Brown, Lower: light brown

9 turbid solution w/ white ppt clear Yellow solution w/ dark Brown

ppt
10 upper layer: light Violet; lower layer: turbid upper layer: blood red liquid
white solution lower layer: clear solution
11 White/milky liquid> Purple dark Red solution
Table 4. Result of Xanthoproteic Test (groups 1-11)

XANTHOPROTEIC TEST

GROUP # INTACT CASEIN ACID/BASE HYDROLYZATE

3 drops HNO3
1 Yellow solution
2 Yellow ppt
3 White ppt in cloudy
solution
4 Yellow spongy mass
at the top
5 Yellow solution
6 light Yellow solution
7 Clear solution w/
White ppt
NaOH 3 drops HNO3 NaOH
red-orange ppt Clear yellow soln Clear orange solution
clear Orange solution clear Orange clear light Yellow solution
solution
dark Yellow Orange clear Orange clear Orange solution
solution w/ dark Red solution
object floating at the
top
Yellow orange solution clear light orange clear light yellow solution
with dusty ppt
dark yellow solution w/ Yellow solution Orange solution w/
orange ppt Orange ppt
Turbid light Yellow light Yellow solution turbid light yellow
solution solution
clear yellow solution w/ Yellow solution >light clear Orange solution
Orange ppt yellow
8 pale Yellow w/ White turbid with White/ light Yellow turbid light yellow
ppt Yellow/Orange ppt solution

9 turbid solution w/ clear Orange solution clear Yellow Orange clear Orange solution
White ppt solution

10 Colorless solution Yellowish solution clear Yellow solution Yellowish Orange w/ ppt

11 White ppt dark Yellow Yellow liquid darker Yellow solution

solution
Table 5. Result of Ninhydrin Test (groups 1-11)

HOPKIN’S-COLE TEST
GROUP ACID/BASE HYDROLYZATE
INTACT CASEIN (interphase)
# (interphase)
1 Light violet Clear liquid

2 Light Violet Light pink

3 Has miniscule amount of Violet in same clear Whitish liquid

clear liquid/ White ppt
4 light Violet White

5 Purple Light Pink

6 light Purple light Pink

7 Light Violet Yellow

8 Violet ring Violet ring

9 clear Violet solution clear solution w/ interphase

10 light Violet Pink

11 clear solution with White ppt interphase

Test: presence of 2 or more peptide bond
Principle: Complexation reaction
(+) Result: Violet solution
(-) Result: Urea & Amino acids
Reagents: 2.5 M NaOH, 0.01 M CuSO4
Color intensity=length of peptide bonds

Figure 1. reaction of copper(II) ion to a peptide

Test: presence of Guanidino group (R)
Principle: Condensation and Complexation
(+) Result: Red coloration (easily disappears)
Reagents: 10% NaOH, 0.02% naphthol solution, 2% NaOBr

Figure 2. Diagram for the Sakaguchi Test

Test: free amino groups (alpha amino group)
Principle: Oxidative Deamination and Condensation
(+) Result: Violet or Deep Blue solution
(when boiled with Ninhydrin)
Reagents: 0.1% Ninhydrin solution (triketohydrindene)

Figure 3. Diagram for the Ninhydrin Test

Test: Aromatic amino acids (F,Y)
Principle: Nitration of Aromatic ring
(+) Result: Yellow ppt > Orange solution
(conc. HNO3) (NaOH)
Reagents: 3 drops conc. HNO3, NaOH

Figure 4. Diagram of
the Xanthoproteic Test
Test: Presence of Indole group (W)
Principle: Complexation and Condensation
(+) Result: Violet interphase
Reagents: 1 mL Hopkin’s-Cole reagent, 2 mL conc. H2SO4

Figure 5. Diagram for Hopkin’s – Cole Test

Table 6. Positive result of each test and group detected

Positive Result Group detected

Biuret Test Violet solution Peptide bonds

Sakaguchi Test Red solution Guanidino group

Ninhydrin Test Violet/Deep-blue Free Amino group

solution

Xanthoproteic Test Yellow precipitate to Aromatic Amino Acid

orange solution

Hopkins-Cole Test Violet interphase Indole Ring

 Incorrect amount of reagents added
 The solution was not allowed to sit still
 Contaminants from the apparatus used
 Shaking of the test tube when it is inclined
 We therefore conclude that the isolated casein contains a

peptide bond with equal or more than two chains from Biuret

test, free Amino group from the protein molecule on Ninhydrin

test, amino acids such as Arginine from Sakaguchi test,

Tryptophan from Hopkins-Cole test, Tyrosine and Phenylalanine

from Xanthoproteic test.

 Bettelheim, F., & Landesberg, J. (2013). Isolation and
identification of Casein. Laboratory experiments
for introduction to general, Organic, and
Biochemistry. 8th ed. pp. 477-479. U.S.A.:
Brooks/Cole,Cengage Learning.
 Cox, M., & Philips, G. (2007). Detection of Amino Acids
and Proteins. Handbook of proteins structure,
functions, and methods. p.212. U.S.A: Wiley and
Sons Ltd.
 Heasley, V., Christensen, V., & Heasley, G. (2013). Proteins
and amino acids. Chemistry and Life in the
Laboratory: Experiments, 6th ed. pp.365-368. USA:
Pearson Education, Inc.
 Japson, R.P., Dr., Manalo, P.B., & Mina, G.E. (2009).
Proteins. Biochemistry: a modular approach.
pp. 110-115 Quezon City: New Day Publishers.
 Definition of Interphase. (n.d.). Retrieved March 04,
2018, from https:// www. chemicool.com/ definition/
interface.html
 Hydrolysis. (2016). Retrieved March 04, 2018, from
https://chem.libretexts.org/Core/Physical_and_Theo
retical_Chemistry/Equilibria/Solubilty/Hydrolysis