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Color Reactions of Intact Protein Casein and Acid/Base Hydrolyzate

Ante, John Michael Cinco, Josemaria Antonio Cruz, Albert Kevin De la Paz, Fallen Grace Gubatan, Glenn

INTRODUCTION

Proteins
formed by two or more amino acids bonded by peptide bonds Roles: structure, enzymes, and hormones etc Proteins can be broken down by chemical or mechanical forces in a process called denaturation

Proteins can be based on structure


Primary, amino acids covalently linked together Secondary, arrangement in space of the atoms in the peptide backbone Tertiary, includes 3D arrangement of all atoms in the protein including the side chains Quaternary, arrangement and interaction between subunits in a protein

Amino Acids
Amino Acids are biological molecules composed of an amino group and a carboxyl group Only 20 are usually found in proteins All amino acids (except glycine) exhibit optical activity due to the presence of an asymmetric Carbon atom.

Amino acids have R-Groups and are classified according to these groups They are based mainly on two criteria namely:
Polarity of side chains or Acidic or Basic side chains

Aside from glycine, most of the amino acids are chiral but are usually found in their L-form

CASEIN
Most important protein in milk making up 80% of the proteins in cow milk and between 20% and 45% of the proteins in human milk. Hydrophobic= poorly soluble in water (H2O)

Acid Hydrolyzate
Tryptophan is destroyed Serine and threonine are decomposed to some extent

Base Hydrolyzate
Racemization Decomposition of Threonine, Serine, Arginine and Cysteine

Color Reactions
functional groups in amino acids and proteins react to produce colored product. color intensity varies in proportion to the number of reacting functional or free groups and their accessibility to reagent.

OBJECTIVE
To characterize acid/base hydrolyzate and intact protein casein using various chemical tests

METHODOLOGY

Color Reaction Test

Dried casein was cut into small pcs.; placed in a mortar

10 ml dist. Water added


Ground until fine soln was obtained

PROTEIN SUSPENSION

HYDROLYZATE

Sakaguchi Test
5 drops of Protein Susp.
+1 drop 10% NaOH +1 drop 0.02% naphthol solution Mix +1 drop 2% NaOBr after 3 min. Note color

5 drops hydrolyzate

Biuret Test
3 drops protein suspension 3 drops hydrolyzate
+1 drop 2.5M NaOH +0.01M CuSO4 Mix Note color

NINHYDRIN TEST
10 drops of Protein Susp.
+1 ml H2O

1 ml of undiluted hydrolyzate

+ 0.5 ml 1% ninhyrdin soln to both


Heated in water bath for 2-3 mins. Note Color

XANTHOPROTEIC TEST
10 drops protein susp. + 1ml H20

1 ml hydrolyzate
+ 3 drops HNO3 Mix Note color Heat 1 min. in water bath Add NaOH dropwise

Test with Litmus paper Note Color

Alkaline Solution

Hopkins Cole Test


2 drops protein suspensio 2 drops Hydrolyzate

+ 2mL of Hopkins-Cole reagent and mix Incline the tube + 2mL of conc. H2SO4 Color of interphase noted

Results

Intact Casein Grp # Biuret Test Sakaguchi test Ninhydrin test Xanthoproteic test HopkinsCole Test

Purple clear solution

Red orange turbid solution

Indigo with violet precipitate

Light yellow ppt. colorless soln


Darker yellow ppt, yellow soln

Purple interphase

Light purple clear solution Violet soln

Red orange clear solution; jellylike Red-orange soln

Light violet solution with light violet ppt Murky soln w/ purple ppt

clear Orange soln Colorless w/ yellow ppt Yellow-orange soln w/ whitish yellow ppt

White

Purple interphase

Purple soln

Orange- red orange soln

Purple soln w/ purple ppt

Small yellow ppt w/ cloudy white soln


Clear orange soln yellow orange ppt

Purple interphase

Light Purple soln

Orange soln

Light purple soln

Clear, colorless w/ suspended particles Orange soln w/ suspended particles

Purple interphase

Intact Casein
Grp # 6 Biuret Test
Purple soln

Sakaguchi test
Orange soln

Ninhydrin test
Clear w/ light violet ppt.

Xanthoproteic test
Clear soln w/ white ppt Red orange ppt

Hopkins-Cole Test
White interphase

Clear Purple soln

Red orange soln

Pale blue-violet soln w/ purple ppt

Turbid soln w/ white particles Orange soln w/ big red& fine white ppt

Light purple interphase

Light blue violet soln

Intesnse redorange, slightly turbid soln

Colorless turbid soln w/ white precipitate

turbid colorless soln

Light purple interphase

turbid yellow soln w/ yellow precipitate

Purple soln

Red-orange soln

Colorless soln w/ white ppt

colorless soln, white ppt Red orange ppt w/ yellow translucent soln

Faint purple interphase

10

Clear light violet soln

Orange soln

Light violet turbid ppt

Turbid colorless soln Turbid orange ppt

Purple interphase

Acid Hydrolyzate
Group # 1 Biuret Test Sakaguchi test Ninhydrin Test maroon clear solution Xanthoproteic test Hopkins-Cole Test Colorless interphase

Blue clear solution

Light yellow clear solution

Bright yellow clear soln Red orang soln

Blue soln

Orange soln

Dark violet/Rusty soln

Bright yellow soln Orange soln

Clear Interphase Colorless turbid interphase Clear soln

Dark gray w/ ppt

Yellow orange soln Orange soln

Maroon/ Yellow soln dark red soln Red orange soln w/ susp. particles Clear brown liquid Clear, light yellow soln Red-orange soln w/ suspended particles

Green-gray soln with particles

light blue soln

Yellow- orange soln

Deep translucent

No change, translucent soln Orange soln w/ red orange ppt.

No interphase

Base Hydrolyzate
Grp # 2 Biuret Test Clear gray soln Sakaguchi test Clear light yellow soln Light yellow soln Ninhydrin Test Dark reddish brown non turbid soln Brown clear soln Xanthoproteic test Yellow soln Dark orange soln Small yellow ppt, cloudy white soln Hopkins-Cole Test Pinkish interphase Light purple interphase

Light purple soln

Yellow-orange ppt and clear orange soln


6 Light yellow soln Light yelloworange soln Dark violet soln Yellowish white soln Red orange & clear soln 8 Light brownish yellow soln Turbid pale yellow soln Clear dark violetdark red soln Darker yellow clear soln Orange yellow clear soln 10 Light brown/yellow solution Yellow orange solution Dark violet soln Clear brownish orange Colorless interphase Light purple interphase Purple interphase

Turbid, yellow ppt

DISCUSSION

Biuret Test
Addition of 2.5M NaOH was needed to form an alkaline solution which is needed for a positive result. Copper(II) ion from CuSO4 reacts with the amide N atoms in a peptide bond Positive result: violet color

Results

O O H N O R H N H Cu
+ 2

H R H

O N H

Results

Sakaguchi Test
presence of arginine Positive result: red-orange to red-colored solution Guanido group in arginine reacts with naphthol in the presence of an oxidizing agent to give off the red color. Condensation reaction

Sakaguchi test
H3 N H3 N
+ +

O O
-

O O NH
-

OH + 2 OH
-

N N

H N

N H

N H2

OH

Sakaguchi Test
Addition of 10% NaOH: catalyzes the reaction Addition of 0.02% naphthol solution: condensation reaction with arginine Addition of 2% NaOBr after 3 minutes: NaOBr and NH in guanido group produces a red complex

Sakaguchi Test
Positive result for Intact casein and acid hydrolyzate. Arginine is present. Base hydrolyzate will give off a light yellow or yellow-orange solution. Alkaline hydrolysis decomposes arginine.

Results

Sakaguchi Test
Possible errors:
Solution will lose its red coloration if left for an extended period of time Errors for Acid hydrolysis: hydrolyzate will require neutralization by addition of a base. If excess base is added, arginine may be destroyed.

Results

NINHYDRIN TEST

detect ammonia or primary and secondary amines free -NH2 group and proteins W/ free amino group reacts with ninhydrin purple-blue complex. Ruhemann's purple = reacts to free amines Most of the amino acids, except Proline, are hydrolyzed and react with ninhydrin *PROLINE GIVES A COLOR YELLOW

Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 the net result is ninhydrin in a partially reduced form hydrindantin:

Ninhydrin then condenses with ammonia and hydrindantin to produces an intensely blue or purple pigment, sometimes called Ruhemann's purple:

PROTEIN SUSPENSION
NINHYDRIN TEST Light violet solution with purple precipitate

ACID HYDROLIZATE
Violet/Dark violet

BASE HYDROLIZATE
Violet/Dark violet

NINHYDRIN TEST group 1 2

Intact Casein

Acid hydrolyzate Positive

Base hydrolyzate

Positive Positive

Positive

3
4 5 6

Positive
Positive Positive Positive

Positive
Positive Positive Positive

7
8 9 10

Positive
Positive Positive Positive

Positive
Positive Negative Positive

oxidative deamination and decarboxylation; reduction of ninhydrin Positive result:


Intact casein: pale violet soln with purple ppt. Base/ Acid Hydrolyzate: violet/ dark violet

Results

Xanthoproteic Test
Tyrosine, Tryptophan, Phenylalanine Electrophilic aromatic substitution Reagents: conc. HNO3, conc. NaOH Positive Result:
Yellow ppt. after addition of conc. HNO3 Orange ppt. after addition of conc. NaOH

Results

O N H3 HNO 3

HO

O N H3 x' cess NaOH O N O


-

O N H3 OH N O
-

Yellow Precipitate

Salt

Results

Grp #

Grp #

INTACT CASEIN
1 Light yellow ppt. colorless soln Darker yellow ppt, yellow soln 2 clear Orange soln 3 Yellow soln Yellow-orange soln w/ whitish yellow ppt 4 Small yellow ppt w/ cloudy white soln Clear orange soln yellow orange ppt 5 Clear, colorless w/ suspended particles Orange soln w/ suspended particles

Clear soln w/ white ppt Red orange ppt

Turbid soln w/ white particles

Orange soln w/ big red & fine white ppt


8

turbid colorless soln turbid yellow soln w/ yellow precipitate

colorless soln, white ppt Red orange ppt w/ yellow translucent soln

10

Turbid colorless soln Turbid orange ppt

Acid Hydrolyzate Group # Group # 1 Xanthoproteic test 2 Bright yellow clear soln

Base Hydrolyzate Xanthoproteic test Yellow soln

Dark orange soln 4 Small yellow ppt, cloudy white soln


Yellow-orange ppt and clear orange soln 6 Yellowish white soln

Red orang soln


3 Bright yellow soln Orange soln 5 Yellow soln Red orange soln w/ susp. particles 7 Clear, light yellow soln Red-orange soln w/ suspended particles 9 Translucent soln, no change Orange soln w/ red orange ppt.

Red orange & clear soln


8 Darker yellow clear soln Orange yellow clear soln 10 Clear brownish orange Turbid, yellow ppt

Hopkins-Cole Test
One of the color reaction tests which is used to detect the presence of Tryptophan in the solution

Tryptophan

protein solution is hydrolyzed by the conc. H2SO4 at the solution interphase The indole group of free Tryptophan reacts with glyoxylic acid to produce purple product

(+) Positive results = violet ring formed in the junction / purple interphase
Protein Acid Base Suspension Hydrolyzate Hydrolyzate HopkinsCole Test
Purple Interphase (+)
Colorless Interphase

(-)

Purple Interphase (+)

Results

Additional Reactions
Paulys Diazol test detection of Tryptophan or Histidine reagent: sulphanilic acid dissolved in hydrochloric acid Forms a diazonium salt The diazonium salt couples with tyrosine or histidine in alkaline medium result: red coloured chromogen (azo dye)

Millons Test
- tyrosine reacts with Millons reagent - Millons rgt: Mercurous nitrate in HNO3 - Positive: flesh to red complex upon heating.

Conclusion
Through various color reactions, intact casein, and base and acid hydrolyzate were known to contain different amino acids as part of their composition.

Reference
Milio, Frank R., & Loffredo, William E. (1995). Qualitative Testing for Amino Acids and Proteins. Modular Laboratory Program in Chemistry Benedict, Stanley R. (1999). A Note on the Preparation of Glyoxylic Acid as a Reagent. The Journal of Biological Chemistry Bettelheim, F., Vandesberg, J., (2013). Laboratory Experiments for Introduction tp General, Organic, and Biochemistry (8th Ed). Brooks/Cole, Cengage Learning: USA. http://amrita.vlab.co.in/?sub=3&brch=63&sim=1094&cnt=1 Retrieved July 5 2013 http://prezi.com/knbu2alx8prs/copy-of-results/ Retrieved July 5 Clark, J.M. Jr., Experimental Biochemistry. 1964. New York: W.H. Freeman and Company. pp. 69-96. Espino-Cabatit, B., Biochemistry, 12e.1988. Philippines: UST Publishing House. pp. 135-138, 141, 142-144