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• The lecture covers protein denaturation, purification, and plasma proteins.

Protein Denaturation

• Protein denaturation refers to the loss of a protein's functional structure, except its primary structure.
• Factors causing denaturation can include heat, pH changes, heavy metals, detergents, mechanical mixing, and digestive enzymes.
• If the cause of denaturation is removed, the protein can sometimes return to its original shape and function through renaturation, although this is uncommon.

Effects of Denaturation

• Denaturation leads to the loss of most bonds, peptide bonds are lost by digestive enzymes only.
• There is decreased solubility and increased viscosity in denaturation.
• Denaturation results in the loss of function; for example, enzymes stop working.
• Clinically, denaturation is relevant in tests like heat coagulation for albumin.
• Misfolded proteins can result in diseases occur (e.g., Alzheimer's, prion diseases).

Plasma Proteins Overview

• Plasma proteins are key soluble components of blood plasma, comprising 60-90 g/L or approximately 4% of the body's total protein.
• Plasma proteins transport substances like hormones and drugs, maintain hemostasis, and provide defense against pathogens.
• The main types of plasma proteins are albumins, globulins, and fibrinogen.
• Albumin is crucial for maintaining osmotic pressure and transport and Globulins are involved in transport and immune functions.
• Fibrinogen is a precursor to fibrin and is essential for blood clotting..
• Proteins can be separated by ammonium sulfate or gel electrophoresis.
• Most plasma proteins, except albumin, are glycoproteins.

Albumin

• The normal plasma level of albumin is 3.4–4.7 g/dL.
• The liver produces about 14 g of albumin daily, contributing to 25% of hepatic protein synthesis.
• Albumin constitutes 60% of total plasma protein and is present both in plasma (40%) and extracellular space (60%).
• Albumin has a half-life of about 20 days
• Albumin is soluble in water, coagulates with heat and can be found in eggs, blood, milk, and cereals.
• Albumin maintains osmotic pressure and provides plasma viscosity.
• It serves as a transport protein for fatty acids, bilirubin, calcium, copper, magnesium, steroid hormones, vitamins, and drugs like penicillin and aspirin.
• Hypoalbuminemia, leads to edema due to cirrhosis, malnutrition, nephrotic syndrome, burns, or malabsorption.
• Hyperalbuminemia, caused by fluid depletion (hemoconcentration).

Globulins

• The normal plasma level of globulins is 2.7-3.2 g/dL.
• Alpha (α1 and α2) and beta (β) globulins are transport lipids, hormones, and trace elements.
• Gamma (γ) globulins provide antibody protection: made by plasma cells and B-cells in the lymphoid system.
• Globulins are insoluble in water but soluble in dilute salt solutions, they also Coagulate by heat and precipitate at half saturation of ammonium sulfate.
• Sources of globulins include egg (egg globulin), blood (serum globulin), milk (lactoglobulin), muscles (myoglobin), and plants (nuts, peas).

Collagen

• Collagen is an insoluble protein that makes up 30% of total body protein.
• Collagen is found in skin, bones, and tendons and other connective tissues.
• it has a composition rich in glycine (33%), proline, and lysine.
• Contains hydroxyproline and hydroxylysine which are formed by post-translational modifications.
• Vitamin C deficiency weakens collagen by preventing hydroxylation, which reduces hydrogen bonding.
• Collagen forms a triple helix and has short helical turns.
• Glycine, the smallest amino acid, allows tight packing.
• Collagen forms a triple helix, allowing tight packing.
• It is stabilized by hydrogen bonds and hydroxyproline.

Elastin

• Elastin is a rubber-like protein that can stretch several times its length and return to its original shape when relaxed.
• Elastin is primarily found in the lungs, walls of large blood vessels, and elastic ligaments.
• Elastin is composed of four interconnected polypeptide chains forming a cyclic structure called Desmosine, which gives elastin its elasticity.
• Consists of 80% hydrophobic amino acids (alanine, valine, leucine, isoleucine) and does not form hydrogen bonds
• Elastin provides flexibility to tissues requiring repetitive stretching and relaxation.

Introduction to Protein Purification

• Protein purification separates a specific protein from others based on properties like solubility, size, charge, or binding affinity.
• Protein purification is essential for diagnostic applications, such as plasma protein electrophoresis, and therapeutic applications (e.g., producing purified proteins for medical use such as insulin).

Salting-In and Salting-Out

• Protein solubility is directly affected by the salt concentration of the solution.
• Salting-In: at low salt concentrations, salts stabilize the charged groups on a protein and attracting water molecules and enhancing protein solubility.
• Salting-Out: at high salt concentrations, salts trap water molecules, reducing their availability to solubilise proteins, resulting in protein precipitation.

Dialysis

• Dialysis separates proteins from small solutes using a semi-permeable membrane.
• The protein solution is placed in a cellophane bag immersed in a solution.
• The pores in the membrane membrane allow water and small molecules to pass through but retain larger protein molecules.
• Most dialysis membranes exclude molecules larger than 3 kDa.

Chromatography- Gel Filtration

• Gel filtration separates proteins based on size using a column filled with porous gel beads.
• Large molecules cannot enter the pores and flow through faster.
• Small molecules enter the pores and move more slowly.
• Applications include estimation of protein molecular weight and desalting protein mixtures.

Ion Exchange Chromatography

• Separates proteins on charge using a column with charged resin.
• Resin carries either negative (polyanionic) or positive (polycationic) groups.
• Oppositely charged proteins bind to the resin at the appropriate pH.
• Proteins are eluted by using a salt solution, which disrupts electrostatic interactions.
• Gradually increasing salt concentration releases weakly bound proteins first, followed by tightly bound proteins.

Purification Techniques Summary

• Salting-In stabilizes proteins through water molecules around charged groups at low salt concentrations, enhancing protein solubility in solutions.
• Salting-Out reduces water by using high salt concentration, causing proteins to precipitate, used to isolate proteins from complex mixtures.
• Dialysis separates proteins from small molecules through semi-permeable membrane to removes salts or impurities from protein solutions.
• Gel Filtration separates by size, where larger molecules elute faster through porous beads useful for molecular weight estimation and desalting.
• Ion-Exchange Chromatography separates by charge using a resin, used to isolate specific proteins based on their charge at a given pH.
• SDS-PAGE denatures proteins separating them by size in an an electric field, useful for determining purity and molecular weight of proteins.