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Chemical Tests for Biomolecules

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  • Manoj Kanth 

Contents (Jump to)

Introduction

Methodology

Results

Discussion

Conclusion

References

Introduction:

Protein is a biomolecule composed of different 20 amino acids and plays a major role in living organisms ( Rangwala, 2008). Protein can be classified into four major categories according to its structure as Primary, Secondary, Tertiary and Quaternary structure (Rangwala, 2008). The structure of a protein differs according to the change in the amino acid it contains and the function of a protein varies with its structure (Buxbaum, 2007). Two types of proteins are there named as globular and fibrous Proteins (Tompa and Fersht 2009). Globular proteins have a tendency to overlap again on themselves into compact spheroid shape where in fibrous proteins the peptide chains are organized in long strands or sheets (Tompa and Fersht, 2009). A protein is functional from its tertiary structure and proteins with similar amino acid sequences frequently have the similar functions (Rangwala, 2008).

Milk plays a major role in a healthy and balanced diet throughout the lifetime as it contains a higher amount of proteins, lipids and minerals (Clemens and Michaelsen, 2015). Casein, lactalbumins and lactoalbumins are the major proteins present in milk (Clemens and Michaelsen, 2015). Casein is highly found in dairy products like milk, yogurt, chocolate and ice cream (Samuels, 2015).

Materials:

Samples

Egg albumin

Milk powder

Regents

Saturated ammonium Sulphate solution

Solid ammonium sulphate

NaOH

CuSO4

Ethanol

Salphasalicylic acid

Lead nitrate

Acetic acid

Water

Acetone

Equipments

Beaker

Bunsen burner

Pipets

Funnels

Methodology:

  1. Precipitation by salt
  1. Half saturation with ammonium sulphate

1 ml of egg albumin was taken and added to equal amount of saturated ammonium sulphate. Then both the sample and reagent were mixed well and kept for 5 minutes. The mixture was filtered using a filter paper. 1 ml of filtrate was taken and 1 ml of NaOH was added to it. Then CuSO4 was added drop wise.

  1. Full saturation with ammonium sulphate

3 ml of egg albumin was taken and solid ammonium sulphate was added until the solution became saturated. The solution was filtered and then 1 ml of the filtrate was taken and 1 ml of NaOH was added. Then CuSO4 was added drop wise.

  1. Precipitation by organic solvents

2 ml of egg albumin was taken and 4 ml of ethanol was added to it. Then the sample and reagents were mixed well and let it stood.

  1. Precipitation by acidic agents

1 ml of egg albumin was taken and equal amount of sulphosalicyclic acid was adeed.

  1. Precipitation by heavy metal ions

1 ml of egg albumin was taken and 5-10 drops of lead nitrate was added.

  1. Precipitation by heat and acid

1 ml of egg albumin was taken in to a test tube and it was held over the flame in standing position. Upper part of the solution was boiled until a cloudy white precipitate was formed. Few drops of 1 % acetic acid was added.

  1. Isolation of casein from milk

17.5 g of non-fat milk powder was taken. 62.5 ml of warm water was added to it in a 400 ml beaker. Then 10 % acetic acid was added drop wise while the temperature was kept at 45 oC. The solution was stirred until it became from milky to a clear solution. Then the solution was filtered and the precipitate was separated. The filtrate was dipped in acetone for 10 minutes and dried well and weighed. Then 1 ml of filtrate was taken and 1 ml of NaOH was added. CuSO4 was added drop wise.

Results:

Test

Observations

Inference

  1. Precipitation by salt
  1. Half saturation with ammonium sulphate

Biurete test for filtrate

Presence of white precipitate

Presence of dark blue colour

Albumin can be precipitated with half saturation

Proteins are absent in the filtrate

  1. Full saturation with ammonium sulphate

Biurete test for filtrate

Presence of white colour precipitate or a clump

Presence of dark blue colour

Globulin can be precipitated using full saturation

Filtrate doesn’t contain protein

  1. Precipitation by organic solvents

Presence of white colour precipitate

Protein can be precipitated by organic solvents

  1. Precipitation by acidic agents

Presence of white colour precipitate

Protein can be precipitated by acidic agents

  1. Precipitation by heavy metal ions

Presence of white colour precipitate

Protein can be precipitated by heavy metal ions

  1. Precipitation by heat and acid

A white cloudy precipitate was observed

Protein can be precipitated by heat and acid

  1. Isolation of casein from milk

Precipitation of casein

15.23 grams

Casein can be precipitated by isolation of milk

Weight of casein precipitated in the end of practical = 15.23 g

Weight of milk powder used for the practical = 17.5 g

Percentage of casein = 15.23/ 17.5 X 100

= 87 %

Half saturation.jpg\Full saturation.jpg

Half saturation with ammonium SulphateFull saturation with ammonium Sulphate

C:\Users\HP\Desktop\B Chem\c) Precipitation by Organic solvents.jpegC:\Users\HP\Desktop\B Chem\d) Precipitation by Acidic agent.jpg

Precipitation by Organic solvents Precipitation by Acidic Agents

C:\Users\HP\Desktop\B Chem\e) Precipitation by Heavy metal ions.jpgC:\Users\HP\Desktop\B Chem\f) Precipitation by Heat & acid.jpg

Precipitation by Heavy metal ions Precipitation by Heat and Acid

C:\Users\HP\Desktop\B Chem\Isolation of Casein 1.jpgC:\Users\HP\Desktop\B Chem\Isolation of Casein.jpg

Isolation of Casein from milk Isolation of Casein from milk

Discussion:

There are two essential made systems for generation of entire casein on a mechanical scale, isoelectric precipitation and rennet coagulation. Casein is regularly utilized as a dietary base for utilization in assessment of vitamins, since it is not difficult to get ready casein as an immaculate protein. Casein is normally ready by corrosive precipitation. Casein is likely exist as micelles in all living organisms. The advancement of free-limit electrophoresis and scientific ultra centrifugation in the 1930’s demonstrated unmistakably that casein and the whey proteins are heterogeneous . While isolating casein from milk the temperature was kept at 45oC to avoid denaturation of milk and the milk would curdle. Adding of Calcium carbonate to the original beaker containing the liquid, neutralized the excess acetic acid and precipitated the inially soluble protein, albumin.

Conclusion:

Casein percentage of the sample was 87 %

Protein can be precipitated by using Salt, Organic solvents, Acidic agents, Heavy metal ions, Heat and acid and isolation methods.

References:

Buxbaum, E. (2007) Fundamentals of Protein Structure and Function. Google books [Online]. Available at: https://books.google.lk/books?id=RxSBnaD6gkYC&printsec=frontcover&dq=protein+structure+and+function&hl=en&sa=X&ei=whtaVc6fH5KdugSczIKAAg&redir_esc=y#v=onepage&q=protein%20structure%20and%20function&f=false (Accessed: 17 May 2015).

Clemens, R., Hernell, O. and Michaelsen, K. (2015) Milk and Milk products in Human Nutrition. [Online]. Nestlenutrition-institute.org. Available at: http://wwwnestlenutrition-institute.org/Resources/Library/Free/workshop/BookNNIW67/Pages/booknniw67.aspx. (Accessed: 18 May 2015).

Rangwala, H., (2008) Protein structure and function prediction using kernel methods. Google books [Online]. Available at: https://books.google.lk/books?id=FjdAznYmkscC&pg=PA10&dq=protein+structure+and+function&hl=en&sa=X&ei=hhtaVdagA86BuwTU9YC4Cw&redir_esc=y#v=onepage&q=protein%20structure%20and%20function&f=false (Accessed: 16 May 2015).

Samuels, M. (2015) Casein Protein Benefits | LIVESTRONG.COM. [Online]. LIVESTRONG.COM. Available at: http://www.livestrong.com/article/86604-casein-protein-benifits/. (Accessed: 16 May 2015).

Tompa, P. and Fersht, A. (2009) Structure and Function of Intrinsically Disordered Proteins. Google books [Online]. Available at: https://books.google.lk/books?id=GzuxFYrzfd4C&printsec=frontcover&dq=protein+structure+and+function&hl=en&sa=X&ei=whtaVc6fH5KdugSczIKAAg&redir_esc=y#v=onepage&q=protein%20structure%20and%20function&f=false (Accessed: 17 May 2015).


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