(Walsh, 2002) stated that, proteins are biological macromolecules composed of amino acids proteins consist of one or more polypeptide which are the chain of amino acids interconnected by peptide bonds.Alberts et al., (2013) detailed that, amino acids of proteins is either hydrophobic or hydrophilic in nature. Therefore the resulting polypeptide chain shows an amphipathic characteristic. Hydrophilic amino acids exist peripherally in some biological system and they are highly water soluble. Whereas some amino acid does not exist the polar groups to the environment.
‘’The most important factors that influence protein solubility are structure, size, charge and the solvent‘’ (Burgess, and Deutscher, 2009). Also Burgess, and Deutscher (2009) stated that, once the precipitation obtained, the solution can be separated by centrifugation or precipitation.
‘’Protein precipitates are aggregates of protein molecular large enough to be visible and to be collected by centrifugation. The distribution of hydrophilic and hydrophobic residues at the surface of a protein determines its solubility properties‘’. (Rosenburg, 2006). Precipitation is mainly done for concentrate the target protein. And it is attained by adding reagents such as salts (ammonium sulfate) or organic solvents (acetone or ethanol). (Hatti-Kaul and Mattiasson, 2003)
1.1.1 Isolation of casein
Milk contains three kinds of proteins: caseins, lactalbumins, and lactoglobulins, all of which are globular proteins. (Spurlock, 2014). Ahluwalia and Dhingra, (2005) stated that, Casein is a combination of phosphoproteins presenting in milk and cheese.it is existing to the amount of 3% in milk along with 4-5% of lactose and 3-4% of fats and the rest is water. Caseins exist in micelles which are composed of sub micelles linked by the characteristic of hydrocolloid which are freely suspended in the aqueous phase of milk. (Tarte, 2009). Casein can be electrophoretically fractioned into four major components: alpha-, beta-, gamma-and kappa- casein. Casein develops precipitation from milk at pH 4.6, which has a negative charge when compare to the pH of the milk. Therefor it can be precipitate as salt by adding acids. (Miller, Jarvis and McBean, 2006).
To learn the methods of protein precipitation and to relate the solubility of protein with its structure.
To learn the methods of isolation of casein from milk and to determine the percentage of casein presented in the (powdered) milk.
- Test tubes
- Filtering paper
- Electronic balance
- Watch glass
- Bunsen burner
- Albumin sample
- Ammonium sulfate
- Sodium hydroxide
- Copper sulfate
- Picric acid
- Lead nitrate
- Powdered milk
- Warm water
1.4.1 Precipitation by salts
Albumin, 3.00ml was taken into a test tube, ammonium sulfate was added to it and was mixed until the solution gets saturated. The solution was allowed to stand for about 5 minutes and filtered by using filter paper. The biuret test was done to the filtered solution. 3.00 ml of filtered solution was taken into another test tube and same amount of NaOH was added to it, CuSO4 was added drop by drop.
1.4.2 Precipitation by organic solvents
Albumin, 1.00 ml was taken into a test tube using a pipette. And 4.00 ml of ethanol was added .the solution was mixed well and was allowed to stand.
1.4.3 Precipitation by acidic agents
Picric acid solution, 1.00 ml was added into 1.00ml of albumin solution.
1.4.4 Precipitation by heavy metal ions
Lead nitrate, 8 drops were added into 1.00 ml of albumin.
1.4.5 Precipitation by heat and acid
Albumin, 10 ml was taken into a test tube and the upper part of the solution was held over the Bunsen flame. After the observation few drops of 1% acetic acid were added.
1.4.6 Isolation of casein
Powdered milk (non-fat), 17.5 g was weighed by using electronic balance and was dissolved by adding 62.5 ml of warm water in a 200ml beaker. Acetic acid (10%) was added in a drop wise manner with stirring until the liquid changes in to clear solution. the obtained solution was filtered by using clamp, filtering material and beaker. The yield casein was allowed to dry and was weighed using electronic balance. Biuret test was done for the filtered solution. 3.00 ml of filtered solution was taken into another test tube and same amount of NaOH was added to it, CuSO4 was added drop by drop.
Precipitation by metal ions
White color precipitation
Proteins can be precipitated by metal ions(positive for proteins)
Precipitation by heat and acid
Initially cloudy white precipitation was observed on the upper part of the solution and by adding acetic acid white color precipitation was observed.
Proteins can be precipitate by heat and acid(positive for proteins)
Precipitation by organic solvents
White color precipitation was observed
Proteins can be precipitate by organic solvents(positive for proteins)
Precipitation by acidic agent
White color precipitation was observed
Proteins can be precipitate by acidic agents. (positive for proteins)
Precipitation of salts
White color precipitation was observed.
Purple color ring was observed
Proteins can be precipitate by salts.
Positive for proteins.
Isolation of casein
Biuret test for filtration
Casein 13.01g was weighed
Purple color ring was formed in filtered casein solution
Yield %= × 100
= × 100
Positive for proteins.
Precipitation of protein can be obtained by isoelectric precipitation method. ‘’isoelectric precipitation is the most widely used method’ (Fox and McSweeney, 2003). Proteins can be precipitated by bringing their pH to its isoelectric point in which protein solubility is very low. (Shankara, 2008)
Proteins can be precipitate by salts in two ways, half saturation with ammonium sulfate and full saturation with ammonium sulfate. Rashmi, (2002) stated that, different proteins show different precipitation reaction towards diverse agents. The full saturation with ammonium sulfate was done in the laboratory. Also the filtrate was tested by biuret reagent, resulted purple color. ‘Compounds with two or more peptide bonds give a violet color with alkaline copper sulfate’ (Rashmi, 2002)
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Proteins are strong in solution when they are enclosed by entirely hydrogen-bonded water molecules, as water molecules with additional hydrogen bonding ability have greater entropy and are more aggressive. (Chaplin, 2014) hydrated sphere decrease the non -polarity. Higher the diameter of the sphere higher the solubility. For an example, it is easy to precipitate globulin from proteins by adding salts, than albumin because globulin has small diameter of hydrated sphere when compare to albumin.
The similar concept is used in precipitating proteins by organic solvents and acidic agents. Organic solvents remove the hydrated sphere and decrease solubility resulting increase precipitation. Acids neutralize the polarity of the hydrated sphere and decrease solubility in order to increase precipitation.
Denaturation occur on heating or adding acidic agents to proteins. Therefor its change the polarity of a protein by changing the arrangements of polar and non-polar groups within the molecule. Less polarity decrease the solubility and increases the precipitation.
Precipitation by heavy metal ions lead nitrate was used instead of lead acetate or mercuric nitrate. Shankara(2008) stated that, metal ions which are positively charged interrelate with negatively charged groups of the protein producing precipitation as metal-proteinate complex.
According to the percentage of yield and from the result of biuret test of the filtrate, there can be some proteins present in the filtrate. Because, the milk contains about 3.5% protein by weight and of the total protein, about 80% is casein and 20% is whey protein. (Miller, Jarvis and McBean, 2006)
Filtration of casein can be done in two ways. Such as, gravitational filtration and sucktional filtration.
Proteins were precipitated by using metal ions, heat, organic solvents, acidic agents and salts.
The percentage of yield casein of the sample is 74.30%.
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