University Of Zimbabwe

Biochemistry Practical

Write-up

Experiment 3: Properties of Proteins

Wednesday 25 September

SAVANHU BEE

R159271A
Title: Investigation of Different Properties of Proteins By Exposing Them To Different

Conditions and Noting Any Visible Changes.

Introduction: Proteins are made up of amino acids. These amino acids are bonded in a

specific sequence which is determined by the section of DNA from which they are

transcribed then translated from. They are bonded by peptide bonds, which is a bond between

the carboxyl group of an amino-acid and the amine group of another amino-acid (Wilbraham

et al, 1987).

The sequence of amino-acids determines the final structure of the protein. The structures are

often described according to the levels primary, secondary and tertiary. These structures give

the proteins specific shape and properties that allow them to serve particular functions e.g.

globular proteins, fibrous proteins, transmembrane proteins and DNA-binding proteins

(Oxtoby and Narchtrieb, 1986).

The primary structure of a protein is the amino-acid sequence that makes up the protein. This

sequence is primarily determined by the DNA and can be modified at various levels before

and after translation. The amino-acid sequence determines the secondary and tertiary

structure of the protein. Genetic defects will therefore affect the amino-acid sequence and

thus the overall structure of the protein (Ebbing, 1996).

The secondary structure of a protein refers to the regular folding of the polypeptide chain into

an alpha-helix or a beta-pleated sheet. The alpha-helix is when the polypeptide chain coils,

forming a helix of 3.6 amino-acids per turn. The –NH groups in one turn form hydrogen

bonds with carbonyl groups in the next. The beta-pleated sheet is when the amino-acids form

a zigzag arrangement. The adjacent chains are held together by hydrogen bonds (Hill and

Feigl, 1978).
The tertiary structure is when the long chains fold into compact, roughly sperical shapes,

forming globular proteins. Most of these proteins are water soluble as they are relatively

small and have hydrophilic surfaces on the outside (Keenan et al, 1980). Several of these

structures can come together to form a quaternary structure. These proteins can also contain a

prosthetic group that can aid in their function.

Proteins can be denatured. This is when it loses its native secondary, tertiary or quaternary

structure. The peptide bonds are not broken by conformational deformation and the denatured

state is always correlated with a loss of enzyme function. In experimental situations, protein

denaturation occurs on addition of urea, guanidine hydrochloride, or detergents that weaken

hydrophobic interactions in proteins and destabilize the native state. Addition of a strong

base, acid, or organic solvent, or heating to temperatures above 60oC are also common ways

to denature a protein (Devlin, 2011).

These experiments were run to investigate the properties of proteins by observing how they

can be precipitated and the reactions of their functional groups.

Materials and Methods: For experiment 1 (a), egg albumin was placed in three test-tubes

and 1 M HCl, 1 M NaOH and water was added to each of the three test-tubes. The tubes were

then boiled for 10 minutes and where neutralized with any visible changes noted. For

experiment 1 (b) a drop of 1% acetic acid was added to a solution of egg albumin and any

visible changes were observed.

For experiment 2, a volume of egg albumin was added to two test tubes, tube A and tube B.

Five drops of 5% NaOH were added to each tube and tube B was boiled for 10 seconds then

cooled. 2% sodium nitroprusside was then added to the two test tube and any visible changes

were observed.
For experiment 3, 95% ethanol was put into two test tubes. 5 drops of plasma were added to

each tube and any visible changes were noted. Water was added to tube A and any visible

changes were observed. Tube B was left to stand for 45 minutes then water was added. Any

visible changes were also noted.

For experiment 4, egg albumin was added to three test-tubes. To one tube, 2% lead acetate

was added, to another 2% silver nitrate was added, then to the last 4% mercuric Chloride was

added. Any visible changes were then observed.

For experiment 5 (a), a few drops of 20% sulphosalicylic acid was added to egg albumin

solution and any visible changes were observed. For 5 (b), Esbach’s solution was added to

egg albumin and any visible changes observed. For 5 (c), 10% trichloracetic acid was added

to egg albumin and any visible change was observed.

For experiment 6 the experiments were carried out with plasma diluted ten times with water.

For 6 (a), saturated ammonium was added to plasma and any visible changes were observed.

Water was then added and any visible change was also observed. For 6 (b), ammonium

sulphate was added to plasma and the solution was mixed then filtered until clear. For 6 (c),

to the filtrate 1% acetic acid was added and any visible changes were observed. Another part

of the filtrate was then saturated with ammonium sulphate then tested for protein (using 1%

acetic acid). Any visible changes were observed.

For experiment 7, the biuret test was carried out on the egg albumin and any visible changes

were observed.

For experiment 8, to some protein in a test-tube concentrated nitric acid was added. This

solution was then boiled until any precipitate formed on addition of acid was dissolved. The
solution was then cooled and 6 M sodium hydroxide was added. Any visible changes were

observed.

For experiment 9, protein solution was placed into two test-tubes, A and B, and 5 M NaOH

was added to each test-tube. Tube A was boiled for 10 seconds and cooled. 1% alpha-

naphthol in alcohol and bromine water were added to each test-tube and any visible changes

were observed.

For experiment 10, five drops of Millon’s reagent were added to the protein solution. The

solution was then heated in a boiling water bath for 10 minutes then cooled to room

temperature. 1% NaNO2 was then added and any visible changes were observed.

Results:

Experiment 1 (a)

Test Tube With 5ml Procedure Observation

Albumin

A 0.5ml 1 M HCl was added White precipitate formed

B 0.5ml 1 M NaOH was added White precipitate formed

C 0.5ml water was added White precipitate formed

Table 1 (a)

Experiment 1 (b)

Volume of Albumin/ml Procedure Observation

3 1 drop 1% Acetic Acid was Thick coagulum was formed

added

Table 1 (b)
Experiment 2

Test tube with Procedure Observation

albumin

A Add 5 drops 5 M NaOH, then add 0.5ml Dark yellow solution

2% sodium nitroprusside solution.

B Add 5 drops 5 M NaOH, boil for 10 Light yellow solution

seconds then cool. Then add 2% sodium
nitroprusside solution.

Table 2

Experiment 3

Test-Tubes with Procedure Observation

95% Ethanol

A Add 5 drops of plasma, then add 10ml Initial precipitate dissolves.

water and mix.

B Add 5 drops of plasma, then leave to Initial precipitate dissolves

stand for 45 minutes and add 10ml of
water.

Table 3

Experiment 4

Test-Tube With 2ml Procedure Observation (1-3 being the order

of 0.5% Egg Albumin of how dense the precipitate was,
Solution 1 being the most dense)

A 3 drops of 2% lead nitrate were Heavy white precipitate formed

added (3)

B 3 drops of 2% silver nitrate was Heavy white precipitate formed

added (1)
 C 3 drops of 4% mercuric chloride was Heavy white precipitate formed
added (2)

Table 4

Experiment 5

Volume of 0.5% egg albumin Procedure Observation

solution/ml
5 Add few drops of 20% White precipitate formed
sulposalicylic acid
3 Add equal volume of Yellow precipitate formed
Esbach’s reagent
3 Add 10% trichloracetic acid White precipitate formed

Table 5

Experiment 6

Volume of plasma/ml Procedure Observations

2ml of diluted plasma Add equal volume of White precipitate formed
saturated ammonium (globulin)
sulphate and filter
Small volume of filtrate Add 1 drop of 1% acetic acid Coagulum is formed
and boil for 10 seconds
Small volume of filtrate Saturate with ammonium Filtrate contains protein
sulphate then test for protein
Table 6

Experiment 7

Volume of 0.5% egg Procedure Observation

albumin/ml
2 Biuret test (add 5 drops 1% White precipitate after
CuSO4 solution followed by volume of copper sulphate is
4ml 5M NaOH) added
Purple solution formed after
 addition of sodium hydroxide
Table 7

Experiment 8

Volume of Protein Procedure Observations

Solution/ml
2-3 Add few drops of Yellow colour deepens to
concentrated nitric acid and orange
heat cautiously to boiling or
until any precipitate formed
on addition of acid is
dissolved. Cool solution and
add 6 M sodium hydroxide in
excess.
Table 8

Experiment 9

Test-Tube containing 3ml of Procedure Observation

protein solution
A Add 1 ml of 5 M NaOH. Mix Brick red colour is formed
and boil contents for 10
seconds and cool. Add 2
drops of 1% alpha-naphthol
in alcohol and 4-5 drops of
bromine water
B Add 1 ml of 5 M NaOH. Add Deeper brick red colour is
2 drops of 1% alpha-naphthol formed
in alcohol and 4-5 drops of
bromine water
Table 9
Experiment 10

Volume of protein Procedure Observation

solution/ml
1 Add 5 drops of Millon’s White precipitate formed on
reagent and heat in water addition of Millon’s reagent
bath for 10 minutes. Cool to White precipitate after
room temperature then add 5 heating
drops of 1% NaNO2 solution. Brick red colour formed after
addition of 1% NaNO2
solution
Table 10

Discussion: For experiment 1 (a) (Table 1 (a)), a white precipitate was formed in all three

test-tubes. This is because the protein was denatured and its structure was distorted, exposing

it hydrophobic regions, hence it was no longer water soluble and it was precipitated. A strong

acid and strong base were added to test-tube A and B respectively and this caused the protein

to denature faster than the protein in test-tube C which just had water. The higher the

temperature is the more energy the atoms making up the protein molecule have so they

vibrate more. These vibrations become large enough to break the bonds holding the protein

together, denaturing it. The strong acid or base also broke some of the bonds in the protein

making it easier to denature. For experiment 1 (b) (Table 1 (b)), a thick coagulum was formed

after the addition of 1% acetic acid. This is because the protein was denatured by the extreme

pH because the acid causes the protein to lose many of its negative charges, leaving it with

positive charges that then repel each other causing the protein to lose its form.

For experiment 2 (Table 2), the protein in test-tube B was denatured to a greater extent than

that in test-tube A since the protein in test-tube B was heated after the addition of alkaline.

This meant that more –SH groups were accessible in test-tube B so a light yellow precipitate
was formed after addition of sodium nitroprusside solution, and a dark yellow precipitate was

formed in test-tube A after the same solution was added.

For experiment 3 (Table 3), white precipitates are formed in both test-tubes initially. This is

because the solubility of the protein decreases with an increase in alcohol concentration. The

precipitate therefore dissolves when water is added to the solution, since the concentration of

alcohol will be decreased.

For experiment 4 (Table 4), the metal ions dissociated from their initial salts and formed an

ionic bond with the protein to form a metal protein salt. The Ag+, Hg+ and Pb2+ cations

reacted with the sulphydryl groups on the proteins. This denatured the proteins since their

structures were broken when these bonds holding them together were broken.

For experiment 5 (Table 5), a white precipitate was formed after sulphosalicylic acid was

added. This is because the protein was precipitated by the presence of an anion that reacted

with a group on the protein, distorting the proteins structure and hence denaturing it. The

same happened when Esbach’s reagent was added and a yellow precipitate was formed and

when trichloracetic acid was added and a white precipitate was formed.

For experiment 6 (a) (Table 6), the protein was salted out by a high concentration of

ammonium sulphate. The protein dissolved when the concentration of ammonium sulphate

was lowered by adding water. For experiment 6 (c), some protein was dissolved in the filtrate

from 6 (b) since the ammonium sulphate was now 50% saturated. This then gave the positive

result for protein when acetic acid was added.

For experiment 7 (Table 7), a purple solution was formed when the copper sulphate and

sodium hydroxide were added to the protein. This is because in the presence of peptides, a

copper(II) ion forms violet-colored coordination complexes in an alkaline solution.

For experiment 8 (Table 8), a yellow colour was formed when nitric acid was added to the

protein. This is because the acid nitrated amino acids carrying aromatic groups (e.g. tyrosine)

forming xanthoproteic acid, which is yellow. It was then heated to dissolve any precipitate.

Alkaline was then added, neutralizing the solution, which turned the yellow colour orange.

For experiment 9 (Table 9), a red colour was produced in the test tubes because the arginine

was able to condense the alpha-naphthol to give a red solution.

For experiment 10 (Table 10), a brick-red colour was formed when Millon’s reagent was

added and this is because it reacts with a tyrosin group on an amino acid. This is a test for

phenolic compounds and not proteins.

Conclusion: Proteins are denatured by high temperatures and an extreme pH. They can aslo

be precipitated by an alcohol, heavy metal cations, anions, ammonium sulphate. The biuret

test is used to test for proteins, the Xanthoproteic reaction is used to test for proteins with a

phenyl group, the Sakaguchi test is used to test for arginine and the Millon’s reaction is used

to test for tyrosine.

: Questions

Proteins

1) are polypeptide are chains with more that 50 amino-acids. The polypeptide is folded

in a particular way which gives it a specific function. Some examples are enzymes,

ion channels and DNA binding proteins.

2) Denaturation is when a protein loses its secondary, tertiary or quaternary structure. It

does not involve breaking the peptide bonds in the primary structure. It can lose this

structure when exposed to extreme pH, high temperatures (>60oC), heavy metals,

organic solvents etc.

References

1) Devlin, T. (2010). Text Book of Biochemistry With Clinical Correlations. 7th edition.

(John Wiley and Sons, Inc, USA) pg 120

2) Ebbing, D. (1996). General Chemistry. 5th edition. (Houghton Mifflin Company,

Dallas, USA) pgs 1069-1070

3) Hill, J and Feigl, D. (1978). Chemistry and Life. (Burgess Publishing Company,

Florida, USA) pgs 506-508

4) Keenan, C Kleinfelter, D and Wood, J. (1980). General College Chemistry. 6th

edition. (Harper and Row Publishers, Inc, New York, USA) pgs 809-815

5) Oxtoby, W Narchtrieb, N. (1987). Principles of Modern Chemistry. (CBS College

Publishing, Tokyo, Japan) pgs 775-779

6) Wilbraham, A Staley, D Simpson, C and Matta, M. (1987). Addison-Wesley

Chemistry. (Addison-Wesley Printing Company, Inc, California, USA) pgs 642-645