1. Proteins

2. Characteristics of proteins:
Are substance of high molecular weight.
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
Serve as structural components of animals
Proteins are polymers consisting of 20 kinds of amino acids.

3. 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.
Catalysis Enzymes catalyze the chemical reactions that allow cells to function.

4. Cont.
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.

5. Amino Acid Amine group acts like a base, tends to be positive.
Carboxyl group acts like an acid, tends to be negative.
“R” group is variable, from 1 atom to 20.
Two amino acids join together to form a dipeptide.
Adjacent carboxyl and amino groups bond together.

6. Peptide Bond Formation
Polypeptides and proteins are held together by amide bonds between the amino-end on one amino acid molecule, and the carboxylate-end of another amino acid molecule. In a peptide, this amide bond is called a peptide bond.
The N-terminus of a peptide/protein is the end with it's alpha-amine NOT involved in a peptide bond.
The C-terminus is the end with its carboxylic acid NOT involved in a peptide bond.

8. Protein Structure
primary structure - lists the amino acids in order from the N-terminus to the C-terminus
secondary structure - describes the "local" folding patterns. Alpha helix and beta sheet are examples.

9. Alpha helix and beta sheet
A) alpha-helix viewed from the side. The hydrogen bonds are shown in green. The hydrogen bonds connect the amido-hydrogens to carbonyl oxygens one
loop of the helix above or below them.
B)beta-sheet - two strands run side-by-side, linked by hydrogen bonds (shown in green). This time the hydrogen bonds connect two strands running parallel to one another.

10. Cont.
tertiary structure - describes how the different elements of secondary structure are arranged in 3-d, which are stabilized by 4 forces:
Ionic bonds, covalent bond (S-S),Van der Waal ,Hydrogen bonds.
quaternary structure - occurs in proteins with more than one polypeptide chain, and describes how the different chains are arranged relative to one another, example : hemoglobin and collagen.

11. Precipitation of Proteins at isoelectric Point
Protein solubility:
There are many factors that contribute to protein solubility. the most important determinant its electrostatic charge. Protein molecules carry charges according to their amino acid sequence and the aqueous solvent pH they're dissolved in.
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.
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 )

12. So protein will be soluble at this pH.
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 isoelectric point(pI).
At a solution pH that is above the pI the surface of the 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.
Repulsive electrostatic force
Repulsive electrostatic forces form when proteins are dissolved in an electrolyte solution. These repulsive forces between proteins prevent aggregation and facilitate dissolution. Upon dissolution in an electrolyte solution, solvent counterions migrate towards charged surface residues on the protein, forming a rigid matrix of counterions on the protein's surface. Next to this layer is another solvation layer that is less rigid and, as one moves away from the protein surface, contains a decreasing concentration of counterions and an increasing concentration of co-ions. The presence of these solvation layers cause the protein to have fewer ionic interactions with other proteins and decreases the likelihood of aggregation. Repulsive electrostatic forces also form when proteins are dissolved in water.

13. The pI of most proteins ranges between the pH 4 to 6.
However, at the pI the negative and positive charges are eliminated, repulsive electrostatic forces are reduced and the dispersive forces predominate. The dispersive forces will cause aggregation and precipitation.
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.
Dispersive or attractive forces exist between proteins through permanent and induced dipoles. For example, basic residues on a protein can have electrostatic interactions with acidic residues on another protein. Therefore, to precipitate or induce accumulation of proteins, the hydration layer around the protein should be reduced. The purpose of the added reagents in protein precipitation is to reduce the hydration layer such as ethanol.

14. 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)

15. Procedure Casein Into a 50 ml volumetric flask add 20 ml of water.
Add 0.25 g of pure casein, followed by the addition of 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.
Casein

16. Setup a series of 9 test tubes.
In the first test tube put 3.2 ml 1 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.

17. 11. Use) +( and )– (signs to describe the turbidity in the different test tubes.
12. You should observe the most precipitation in the test tube which has the pH around 4.7 (close to the isoelectric point of casein).
Tube 1 2 3 4 5 6 7 8 9 In
CH3COOH
1.6
0.8
0.4
0.2
0.1
0.05
0.025
0.012
0.006 pH
3.5
3.8
4.1
4.4
4.7
5.0
5.3
5.6
5.9
Turbidity