K.

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Clayden, J., Greeves, N., Warren, S., & Wothers, P. (2000). Organic
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Jurusan Kimia FMIPA Unesa.
Kristinsson, H. G. and Rasco, B. A. 2000. Fish Protein Hydrolysates:
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L. ATTACHMENT
1. QUESTIONS ANSWER
1) Explain what is the function of testing the protein with each test reagent
(CuSO4, HgCl2, HNO3, Pb-acetate)!
Answer:
The function of protein test with each reagent as follows:
‐ CuSO4 is used to test for the presence of heavy metals in proteins
characterized by presence of precipitation when positive proteins
contain heavy metals.
‐ HgCl2 is used for the test of a protein containing a phenyl hydroxyl
group (-OH).
‐ HNO3 is used to test for the presence of benzene rings from the amino
acid salt of the proteins, ie in this experiment when concentrated nitric
acid is added and produces nitrobenzene derivatives.
‐ Acetate pb is used to test for the presence of cysteine and methionine
amino acids, which in this experiment will produce a black solution
because the S atom reacts with acetic acid to form PbS precipitate.
2) How does the effect of organic solvents (acetone and ethanol) on the
nature of protein denaturation?
Answer:
The effect of organic solvents (acetone, ethanol) on the nature of protein
denaturation is that proteins or nucleic acids will lose their secondary and
tertiary structures because organic solvents result in denatured proteins.
3) List the various bonds that cause the polypeptide to be stable in alpha-
helical form!
Answer:
‐ Disulfide bond
Formed between 2 cysteine residues interconnected 2 parts of the
polypeptide chain through cysteine residues.

Page | 61
 ‐ Hydrogen bond
Formed between the NH- or -OH groups and the C = O groups in the
peptide or -COO-bond in the R group.

Page | 62
DOCUMENTATION
1. Protein denaturation
a. Denaturation because of addition acetic acid
5 mL protein solution Test tube I : white
-5 mL of milk (I) solution
-5 mL of egg solution (II) Test tube II: yellowish
(egg white dilute with solution
water in ration 1:3)
1. Entered into test tube I
and II

2. Added 2 drops acetic Formed flakes in both

acid 1N while shaking solution

3. Heated in water bath

in 5 minutes
4. Observed the changes
in precipitation

Page | 63
Result for milk solution Formed precipitate

Result for egg solution Formed precipitate

b. Denaturation causing heating

3 mL protein solution Test tube I: yellowish
-3 mL of egg (I) solution
solution (egg Test tube II: white
white dilute with solution
water in ratio 1:3)
-3 mL of milk solution
(II)
1. Entered into rest tube
I and II
2. Heated for 1 minute

Result Flake precipitate occur

3. Divided into 2 test in both solution
tube for each sample

Page | 64
Milk solution after Parts I: Protein
Parts I precipitate
4. 2 drops of (NH4)SO4 Parts II: Protein
solution precipitate (more clear)
5. Heated
Parts II
4. Heated

Egg solution after Parts I: Protein

Parts I precipitate
4. 2 drops of (NH4)SO4 Parts II: Protein
solution precipitate (more clear)
5. Heated
Parts II
4. Heated

c. Denaturation because addition of formaldehyde

1,5 mL formaldehyde Colorless solution in both
1. Entered into test tube I test tube
and II

2. Added protein Test tube I: Formed

solution drops by white precipitate
drops Test tube II: Formed
-egg solution (I) white precipitate
(egg white dilute
with water in ratio
1:3)
-milk solution (II)

Page | 65
2. Ampotheric properties of protein
a. For amphoteric protein test (from egg)
3 mL of aquadest Colorless solution in both
1. Entered into test tube I test tube
and II
2. Added 2 drops of HCl

3. Added 3 drops of Purplish blue solution

cango indicator

4. Added 2 mL protein Test tube I: pink solution

solution with precipitate
-milk solution (I) Test tube II: orange
-egg solution (II) (eqq solution
white dilute with water in
ration 1:2)

b. Test with alkaline atmosphere

 Test tube I
3 mL NaOH 0,1 M Colorless solution
1. Entered into test tube

2. Added few drops of Pink solution

PP indicator

Page | 66
  Test tube II
3 mL protein solution Test tube I : yellowish
-3 mL of egg solution (I) solution
(egg white dilute with Test tube II: white
water in ratio 1:2) solution
-3 mL of milk solution
(II)
1. Entered into test tube
2. NaOH 0,1 M solution Egg solution: pink clear
from test tube I solution
Milk solution: pink
solution

 Test tube III

3mL NaOH 40% solution Colorless solution
1. Entered into test tube

2. Added few drops of Pink solution

PP indicator

 Test tube IV
3 mL protein solution Test tube I : yellowish
-3 mL of egg solution (I) solution
(egg white dilute with Test tube II: white
water in ratio 1:2) solution
-3 mL of milk solution
(II)
1. Entered into test tube

Page | 67
2. NaOH solution from -Egg solution: pink clear
test tube III solution
-Milk solution: pink
solution

3. Precipitation of protein
a. Precipitation of protein with Amonium Sulfate
3 mL protein solution Test tube I : yellowish
-3 mL of egg solution (I) solution
(egg white dilute with Test tube II: white
water in ratio 1:2) solution
-3 mL of milk solution
(II)
1. Entered into test tube
2. Added 3-4 mL Turbid solution in both
saturate solution of test tube
ammonia sulphate
3. Shake

4. 2-3 mL aquadest Dissolve precipitation in

5. Shake both solution

b. Precipitation of protein with mineral acid

 Test tube I
1 mL of HNO3 Colorless solution
concentrated
1. Entered into test tube

Page | 68
2. Added 1-1,5 mL White ring formed as a
protein solution protein precipitate
-3 mL of egg solution (II)
(egg white dilute with
water in ratio 1:2)
-3 mL of milk solution (I)

3. Added 1 mL of HNO3 More precipitate

 Test tube II
1 mL of HNO3 Colorless solution
concentrated
1. Entered into test tube

2. Added 1-1,5 mL White ring formed as a

protein solution protein precipitate
-3 mL of egg solution (II)
(egg white dilute with
water in ratio 1:2)
-3 mL of milk solution (I)

3. Added 1mL of HCl More precipitate

c. Precipitation of protein with heavy metal

 With CuSO4 (Test tube I)

Page | 69
1mL of protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

2. Added CuSO4 drop by Blue precipitate in both

drop test tube
3. Shake

4. Added CuSO4 0,1 M Dissolve precipitation in

both test tube

 With PbSO4 (Test tube II)

1mL of protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

2. Added PbSO4 drop by White precipitate in both

drop test tube
3. Shake

Page | 70
4. Added PbSO4 0,1 M Dissolve precipitation in
both test tube

 With ZnSO4 (Test tube III)

1mL of protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

2. Added ZnSO4 drop by White precipitate in both

drop test tube
3. Shake

4. Added ZnSO4 0,1 M Dissolve precipitation in

both test tube

 With FeSO4 (Test tube IV)

1mL of protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

Page | 71
2. Added FeSO4 drop by Yellow precipitate in
drop both test tube
3. Shake

4. Added FeSO4 0,1 M Dissolve precipitation in

both test tube

4. Protein color reaction

a. Biuret reaction
1,5 mL protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

2. Added 1 mL NaOH Purple solution in both

40% test tube
3. Added drops of
CuSO4 0,5%

b. Chantoprotein solution
1,5 mL protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

Page | 72
2. Added 1 mL HNO3 Yellow solution in both
3. Heated test tube

4. Cooled Orang solution in both

5. Added ammonia test tube

c. Ninhydrin reaction
1,5 mL protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube
2. Set pH until 7 (+water)
3. Added 10 drops
ninhydrin solution 0,2%

4. Heated at 100℃ for

10 minutes

Result Color changes into:

-milk solution: blue
solution
-egg solution: clear
purple soution

d. Millon reaction

Page | 73
2 mL protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube

2. Added 1 mL millon white solution in both

reagent test tube

3. Heated

Result after first heating Milk solution (right):

yellow precipitate
-Egg solution (left):
yellow precipitate

4. Cooled with water Milk solution (left):

5. Added drops of yellow precipitate ++
NaNO3 1% -Egg solution (right):
6. Heated again yellow precipitate ++

e. Hopke-cole reaction

Page | 74
1,5 mL protein solution Test tube II : yellowish
-Milk solution (I) solution
-Egg solution (II) (egg Test tube I: white
white dilute with water in solution
ratio 1:2)
1. Entered into test tube
2. added 1 drop of dilute
formaldehyde
3. Added 1 drop of
HgSO4
4. 1 mL concentrated formed 2 layers (in the
sulfuric acid boundary filed there is a
purole ring) in both of
test tube

5. Protein hydrolysis and sulphur test

1mL of protein solution Test tube I : yellowish
-Milk solution (II) solution
-Egg solution (I) (egg Test tube II: white
white dilute with water solution
in ratio 1:2)
1. Entered into test tube

2. Added 1 drop of -Milk solution: orange

NaOH 40% solution
3. heataed for 1 -Egg solution: yellow
minutes: solution

4. 1 drop of Pb-acetate Both accur black

precipitate (PbS)

Page | 75