Precipitation of Proteins at isoelectric Point

1. Precipitation of Proteins at isoelectric Point

2. Objectives: • General information about proteins . • To understand the Isoelectric Point . • To find the PI of Casein protein .

3. Proteins • Proteins are polymers consisting of 20 kinds of amino acids. • Are substance of high molecular weight from 5000 to1000,000 daltons. • All protein Contain C, H, O, N, and most contain sulfur, some contain phosphorus and a few have mineral elements such as Fe, Mg and Cu.

4. Proteins

5. Protein functions • Structure some proteins provide structural support collagen, hair, crystallins(eyes). • Transport some proteins are responsible for the transportation of smaller molecules from one part of the body to another, transport across cell membranes, etc . An example is hemoglobin, which transports oxygen from the lungs to cells throughout the body.

6. Catalysis enzymes catalyze the chemical reactions that allow cells to function. • Storage Myoglobin is an example of a storage protein. Myoglobin stores oxygen in muscles so that during exercise a ready supply of oxygen is available in the muscle tissue. • Hormones some hormones are proteins, insulin is an example. Hormones serve as chemical messengers, carrying signals from one part of the body to another.

7. Protein structure • Primary structure • Secondary structure • Tertiary structure • Quaternary structure

8. Introduction • Properties of amino acids in proteins and peptides are determined by the R group . • Important to know which groups on peptides and proteins will be protonated at a certain pH. • Protein molecules carry charges according to their amino acid sequence and the aqueous solvent PH they’re dissolved in.

9. Precipitation of Proteins at isoelectric PointProtein solubility • There are many factors that contribute to protein solubility. • The most important determinant its electrostatic charge. • 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. • Hydrophilic amino acid like (Arginine, Asparagine, Aspartate, Glutamine, Glutamate, Histidine, Lysine, Serine and Threonine) • hydrophobic amino acid (Valine, Tyrosine, Tryptophan, Proline, Phenylalanine, Methionine, Leucine, Isoleucine, Cysteine and Alanine )

10. Charged and polar surface residues interact with ionic groups in the solvent and increase solubility. • The net charge of a protein molecule is the arithmetic average of all charges. At a certain solvent PH the protein net charge will be zero this is called the isoelectirc point. • At a solution PH that is above the PI the surface of protein is predominantly negatively charged and therefore like charged molecules will exhibit repulsive forces. • Likewise the surface of the protein is predominantly positively charged at a solution PH that is below the PI, and repulsion between proteins occurs, so protein will be soluble at this PH.

11. However, at the PI the negative and positive charges are eliminated, repulsive electrostatic forces are reduced and the dispersive forces will cause aggregation and precipitation. • The PI of most proteins ranges between the PH 4 to 6. • 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.

12. 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)} 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

13. Notes • Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. • Proteins have different pI because of their different amino acid sequences (i.e.,relative numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution. When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in solution.

14. Experiment

15. precipitation of proteins 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.

16. Setup a series of 9 test tubes to identify the best tube that have the most precipitate PI . • 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).

17. 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).

19. Results

20. Results Sheet • Results: • PKa = 4.5 • Use the following to indicate the precipitate: • - no precipitate • + few ppt • + + Moderate ppt • + + + maximum ppt

21. Comment your results : • in tubes 1.2: • in tubes 3.4.5.6 : • in tube 7: