Experiment 2 -

color reactions of Proteins .

1) Biuret reaction

AIkalinesolutionofproteintreatedwithcuSILP.RO
-

sepinKtoui¥!dIue
oletcolorationdependi#onthelengthI forms
to
a
the
colored
peptide Bond ; Cudi) binds with
complex .
N and

Can also occur if 2

Carbonyl ( R co R ) groups are attached by nitrogen / carbon
-
-

Good for determining the extent protein hydrolysis

-

or .

Smaller peptides and amino acids show no coloration .

1mL 2% Albumin
1mL 10% NaOH

ftp.
0.5% CuSO4 Peptone = Rose pink

Albumin = Violet
2mL 2% Peptone
1mL 10% NaOH
0.5% CuSO4

Before CuSO4 After CuSO4

2) Xanthoproteic reaction

proteinstreatedwithconc.HN#

↳ Turn yellow but can be neutralized with alkali NH4 OH

-

Good in ↳
⑧
reaction
-06ft
-

Due to of benzene
.

the nitration ring

an activated benzene
Amino acids Tyrosine Tryptophan Phenylalanine
-

electron Ht ) :

( donates
,
.

ring

1mL 2% Albumin
O.5mL Conc. HNO3
NH4OH
ftp. ftp.
Nor

it
⇐ Albumin + HNO3 Albumin + HNO3
(Heated)
Cooled and NH4OH
was added.
Orange ppt.
(nitrobenzene) difficult to nitrate under normal condition
→
phenylalanine is .
 3) Millon 's test

lvlillorisreagentaddedtoaprotcinsolufiontvaa
-

4. preoipitatesasmercury
mercuryBHNOQ 47 when flesh red color if

↳
.
heated turns a to
•
mercurous nitrates protein contains tyrosine
•
mercuric nitrates
when heated turns flesh to red color
Hg Nz 06
a

because of the Phenol group

1mL 2% Albumin
⑧O
Million’s Reagent
(1 Hg. : 2 Conc. HNO3)

Ltd -

mercury
phenolate .

Millon’s Reagent Mixture was heated

+ Albumin Red Color
White Jelly Ppt.

4) Hopkins -

Cole :
Glyoxylic acid reaction

glyoxylicaa.de/CHOC0OH)andHzS0aaddedtoproteinsoluti#
-

t wo ↳ violet ring is produced at the point of contact

• reduction of of
-
the two solutions .

Oxalic acid
↳
with magnesium
powder
/ Due
in
to
the
the
presence
tryptophan
of
component
an
.
indole nucleus .

Oxo acetic acid .

Liz Tryptophan condenses with the aldehyde

1mL 2% Albumin
1mL Hopkins-Cole Reagent
1mL pure conc. H2SO4

Hzso . was used +. ①

t
Violet Ring
Faint due to quality of
reagents
"*"
.

O O
hydrolyse the tryptophan 11 11
free to H C C OH
so it is -
-
-

react with
acid tryptophan will be connected
glyoxylic 2 ,

by the glyoxylic acid (aldehyde part)

5) Heller 's ring test

HNOzisinthebottomlayerandasolutionofprotein-isad.de#
-

4. white precipitate of coagulated protein at the junction of

thetwo.layers.lt
Coagulation due to minerals like Hd ,
HMOs
↳ acids
inorganic .

5mL Conc. HNO3

2% Albumin

Stamm

White ppt. form in the Turns yellow after standing

interface after contact

6) Reduced Sulfur Test

Protein solution is heated with NaOH and Pb ( CHS COO) 2 is added

Is
↳
Blackleadsulfideprecipitateformsl
"
protein som .

containing sulfur . when heated with NaOH

splits up the sulfur to form Naas and it forms

a black precipitate with Pb ( CH 300072 .

Amino acids with sulfur : cystine , cysteine ,

methionine .

2mL 2% Albumin
2mL 40% NaOH
10 drops Pb(CH3COO)2

Stamm

There is ppt. of black precipitate

The solution turned black
after heating for 2 minutes
7) Adam kiewicz reaction :
Hopkins -

Cole

Protein solution is added with glacial acetic acid .

and H2S 04
isslowlyadded.to/-hesideofthetub#
↳ violet ring is produced at the point of contact

ofthetwosolutions.lt
/ Due
in the
to the
presence
tryptophan
of
component
an
.
indole nucleus .

↳• Tryptophan condenses with the aldehyde

Glacial Acetic Acid is Anhydrous acetic acid ; when exposed to

iii. t.it.iiiiiiii.iiiiiiiiiiiro.im
"
"

. oiea
.

3 drops 2% Albumin
5mL Glacial CH3COOH
Conc. H2SO4
6- Should be presence
of violet ring

Stand for a few minutes