What is protein: • Proteins are key players in our living systems. • Proteins are polymers consisting of 20 kinds of amino acids. • with mol.wt from 5000 to1000,000 daltons. • N is most distinguished element: among the composing elements of C,H, N, O, S.
Peptide Bond Formation • Each Individual amino acids form a polypeptide chain • rigid
nature of protein structure Primary structure(Amino acid sequence) ↓ Secondary structure （α-helix, β-sheet） ↓ Tertiary structure Three-dimensional structure formed by assembly of secondary structures ↓ Quaternary structure Structure formed by more than one polypeptide chains
Precipitation of Proteins at isoelectric Point • Protein solubility: • The solubility of proteins in aqueous buffers depends on the distribution of hydrophilic and hydrophobic amino acid residues on the protein’s surface. • Proteins that have high hydrophobic amino acid content on the surface have low solubility in an aqueous solvent. • Charged and polar surface residues interact with ionic groups in the solvent and increase solubility.
Sine the solubility of protein such as casein is not affected by heat because it does not contain disulphide bonds and lack the tertiary structure. • The solubility of casein depends greatly on the PH of the medium. • The intermediate PH at which a protein molecule has a charge of zero is called, the isoelectric point of that protein. • At this point the solubility of protein is minimum, but increases with increasing acidity or alkalinity
maximum precipitation can be obtained at the isoelectric point by addition of some reagents such as, ethanol which dehydrates the molecule and allow neutralization of charge. • The pI of most proteins ranges between the pH 4 to 6. • When microorganisms grow in milk, they often produce acids and lower the pH of the milk. • The phenomenon of precipitation or coagulation of milk protein (casein) at low pH as milk becomes spoiled is one of the common examples of protein isolation due to changes in the pH.
Isoelectric point (pI):is the pH-value of a solution at which the total net charge of a protein equals zero. • At a solution pH that is above the pI the surface of the protein is predominantly negatively charged • Likewise the surface of the protein is predominantly positively charged at a solution pH that is below the pI,
Principle: • Using acetate buffer of different PH values to find the isoelectric point of casein • can be obtained by determining the PH where minimum solubility. • The PH of any solution can be calculated from Handersonhasselbalch • equation. • PH = Pka + log (casein acetate sodium ) • (acetic acid)]
Procedure • Into a 50 ml volumetric flask add 20 ml of water. • Add 0.25 g of pure casein, followed by the addition of 5 ml of 1 N NaOH solution. • Once casein is dissolved, add 5 ml of 1 N acetic acid solution, then dilute with H2O to 50 ml and mix well. The resulted solution is a 0.1 N casein acetate sodium.
Setup a series of 9 test tubes. • In the first test tube put 3.2 ml1 N CH3COOH, and 6.8 ml H2O and mix thoroughly. • In each of the other test tubes (2-9) put 5 ml H2Od. • From the test tube 1 transfer 5 ml to the test tube 2, and mix thoroughly. • Repeat step 7 for the rest of test tubes (3 - 9).
Now to each test tube (1 -9) add 1 ml of the casein acetate sodium solution, and shake the test tubes immediately. • Let the samples stand for 30 min, and note the turbidity in the 9 test tubes. • Use) +( and )– (signs to describe the turbidity in the different test tubes. • You should observe the most precipitation in the test tube which has the pH around 4.7 (close to the isoelectric point of casein).
Results Sheet • Results: • PKa = 4.5 • Use the following to indicate the precipitate: • - no precipitate • + few ppt • + + Moderate ppt • + + + maximum ppt
Comment your results : • in tubes 1.2: • in tubes 184.108.40.206 : • in tube 7: