University Of Zimbabwe
Biochemistry Practical Write-up
Experiment 3: Properties of Proteins
Wednesday 25 September
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.
Experiment 1 (a)
Test Tube With 5ml Albumin Procedure Observation
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 added Thick coagulum was formed
Table 1 (b)
Test tube with albumin Procedure
A Add 5 drops 5 M NaOH, then add 0.5ml 2% sodium nitroprusside solution. Dark yellow solution
B Add 5 drops 5 M NaOH, boil for 10 seconds then cool. Then add 2% sodium nitroprusside solution. Light yellow solution
Test-Tubes with 95% Ethanol Procedure Observation
A Add 5 drops of plasma, then add 10ml water and mix. Initial precipitate dissolves.
B Add 5 drops of plasma, then leave to stand for 45 minutes and add 10ml of water. Initial precipitate dissolves
Test-Tube With 2ml of 0.5% Egg Albumin Solution Procedure Observation (1-3 being the order of how dense the precipitate was, 1 being the most dense)
A 3 drops of 2% lead nitrate were added Heavy white precipitate formed (3)
B 3 drops of 2% silver nitrate was added Heavy white precipitate formed (1)
C 3 drops of 4% mercuric chloride was added Heavy white precipitate formed (2)
Volume of 0.5% egg albumin solution/ml Procedure Observation
5 Add few drops of 20% sulposalicylic acid White precipitate formed
3 Add equal volume of Esbach’s reagent Yellow precipitate formed
3 Add 10% trichloracetic acid
White precipitate formed
Volume of plasma/ml Procedure Observations
2ml of diluted plasma Add equal volume of saturated ammonium sulphate and filter White precipitate formed (globulin)
Small volume of filtrate Add 1 drop of 1% acetic acid and boil for 10 seconds Coagulum is formed
Small volume of filtrate Saturate with ammonium sulphate then test for protein Filtrate contains protein
Volume of 0.5% egg albumin/ml Procedure Observation
2 Biuret test (add 5 drops 1% CuSO4 solution followed by 4ml 5M NaOH) White precipitate after volume of copper sulphate is added
Purple solution formed after addition of sodium hydroxide
Volume of Protein Solution/ml Procedure Observations
2-3 Add few drops of concentrated nitric acid and 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. Yellow colour deepens to orange
Test-Tube containing 3ml of protein solution Procedure Observation
A Add 1 ml of 5 M NaOH. Mix and boil contents for 10 seconds and cool. Add 2 drops of 1% alpha-naphthol in alcohol and 4-5 drops of bromine water Brick red colour is formed
B Add 1 ml of 5 M NaOH. Add 2 drops of 1% alpha-naphthol in alcohol and 4-5 drops of bromine water Deeper brick red colour is formed
Volume of protein solution/ml Procedure Observation
1 Add 5 drops of Millon’s reagent and heat in water bath for 10 minutes. Cool to room temperature then add 5 drops of 1% NaNO2 solution. White precipitate formed on addition of Millon’s reagent
White precipitate after heating
Brick red colour formed after addition of 1% NaNO2 solution
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.
Proteins are polypeptide 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.
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.
Devlin, T. (2010). Text Book of Biochemistry With Clinical Correlations. 7th edition. (John Wiley and Sons, Inc, USA) pg 120
Ebbing, D. (1996). General Chemistry. 5th edition. (Houghton Mifflin Company, Dallas, USA) pgs 1069-1070
Hill, J and Feigl, D. (1978). Chemistry and Life. (Burgess Publishing Company, Florida, USA) pgs 506-508
Keenan, C Kleinfelter, D and Wood, J. (1980). General College Chemistry. 6th edition. (Harper and Row Publishers, Inc, New York, USA) pgs 809-815
Oxtoby, W Narchtrieb, N. (1987). Principles of Modern Chemistry. (CBS College Publishing, Tokyo, Japan) pgs 775-779
Wilbraham, A Staley, D Simpson, C and Matta, M. (1987). Addison-Wesley Chemistry. (Addison-Wesley Printing Company, Inc, California, USA) pgs 642-645