Protein Denaturation and Purification PDF

1/20/2025 The Protein denaturation, purification and plasma proteins Dr. Lujain Almohammadi Outline Protein Denaturation Definition: What denaturation and purification mean. Causes: Factors that lead to protein denaturation (e.g., heat, p...

1/20/2025 The Protein denaturation, purification and plasma proteins Dr. Lujain Almohammadi Outline Protein Denaturation Definition: What denaturation and purification mean. Causes: Factors that lead to protein denaturation (e.g., heat, pH). Effects: Chemical, physical, and biological changes. Clinical Relevance: Examples and related diseases. Protein Purification Importance: Why purification matters in science and medicine. Techniques: Salting-In and Salting-Out, Dialysis, Chromatography and SDS-PAGE. Plasma Proteins Overview: Key roles of plasma proteins in the body. Types: Albumin, Globulins, Fibrinogen. Clinical Relevance: Conditions like hypoalbuminemia and their impact. Special Proteins Collagen: Structure, importance in connective tissue, clinical relevance. Elastin: Stretchable properties, role in lungs and arteries. 1  1/20/2025 What is Protein Denaturation? Definition: Loss of protein’s functional structure (except primary). Causes: Heat. pH. Heavy metals. Detergents. Mechanical mixing. Digestive enzymes Re-naturation: When the cause of denaturation is removed, the protein can return to its original shape and function (this is not common). Effects of Denaturation Chemical: Loss of most bonds, peptide bonds are lost by the effect of digestive enzymes only. Physical: Decreased solubility, increased viscosity. Biological: Loss of function (e.g., enzymes stop working). Clinical Relevance of Denaturation Examples: Heat coagulation test for albumin. Some Diseases result from misfolded proteins (e.g., Alzheimer’s, prion diseases). 2  1/20/2025 Plasma Proteins Overview Plasma proteins are the key soluble components of blood plasma, making up 60-90 g/L (about 4% of the body’s total protein). Functions: Transport substances (e.g., hormones, drugs). Maintain hemostasis (blood clotting). Provide defence against pathogens (antibodies). Main Types: Albumins: Major protein for maintaining osmotic pressure and transport. Globulins (α1, α2, β, γ): Transport and immune functions. Fibrinogen: Precursor to fibrin, essential for blood clotting. Separation: Proteins can be separated by ammonium sulfate or gel electrophoresis. Glycoproteins: Most plasma proteins (except albumin) are glycoproteins, meaning they contain carbohydrate groups. Albumin Albumin (Plasma Normal Level: 3.4–4.7 g/dL) Production: The liver produces ~14 g/day, contributing to 25% of hepatic protein synthesis. Key Features: Major plasma protein (60% of total plasma protein). Present in plasma (40%) and extracellular space (60%). Half-life: ~20 days. Soluble in water; coagulated by heat. Found in: eggs, blood, milk, and cereals. 3  1/20/2025 Albumin Functions: 1. Maintains osmotic pressure and provides plasma viscosity. 2. Serves as a transport protein for: Fatty acids, bilirubin. Calcium, copper, magnesium. Steroid hormones, vitamins, and drugs (e.g., penicillin, aspirin). Clinical Conditions: Hypoalbuminemia (Low Levels): Causes: Cirrhosis (liver disease), malnutrition, nephrotic syndrome, burns, malabsorption. Effect: Edema (fluid accumulation). Hyperalbuminemia (High Levels): Cause: Fluid depletion (hemoconcentration). Globulins Globulins (Plasma Normal Level: 2.7–3.2 g/dL) Functions: 1. α-Globulins (α1 and α2) and β-Globulins : Transport lipids, hormones, and trace elements; produced in the liver. 2. γ-Globulins: Protective function as antibodies; made by plasma cells and B-cells in the lymphoid system. Properties: 1. Insoluble in water but soluble in dilute salt solutions. 2. Coagulate by heat. 3. Precipitate at half saturation of ammonium sulfate. Sources: Egg (egg globulin), Blood (serum globulin), Milk (lactoglobulin), Muscles (myoglobin). Plants (nuts, peas). 4  1/20/2025 Collagen and Elastin (Special Proteins) Collagen is an insoluble protein that makes up 30% of total body protein. Found in connective tissues like skin, bones, and tendons. Composition: Rich in glycine (33%), proline, and lysine. Contains hydroxyproline and hydroxylysine formed by post-translational modifications. Vitamin C deficiency weakens collagen by preventing hydroxylation, reducing hydrogen bonding. Structure: Collagen forms a triple helix (three polypeptide chains are wound together). It has a firm structure due to: 1. Short helical turns (3 amino acids per turn). 2. Glycine (smallest amino acid) allows tight packing. 3. Assembly into fibers. 4. Chemical cross-linking 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. It is found in the lungs, walls of large blood vessels, and elastic ligaments. Stretchability: Composed of four interconnected polypeptide chains forming a cyclic structure called Desmosine, which gives elastin its elasticity. 80% hydrophobic amino acids (alanine, valine, leucine, isoleucine): These do not form hydrogen bonds, allowing the elastin core to stretch and recoil easily. Function: Provides flexibility to tissues that require repetitive stretching and relaxation. 5  1/20/2025 Introduction to Protein Purification Protein purification isolates a specific protein from others using methods based on properties like solubility, size, charge, or binding affinity. It is essential for diagnostic (e.g., 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, attracting water molecules and enhancing protein solubility. Salting-Out: At high salt concentrations, salts trap water molecules, reducing their availability to solubilise proteins. This results in protein precipitation. 6  1/20/2025 Dialysis Dialysis separates proteins from small solutes using a semi-permeable membrane. The protein solution is placed in a cellophane bag, which is immersed in a solution. Process: The pores in the membrane allow water and small molecules to pass through but retain larger protein molecules. Membrane Size: 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. Process: Large molecules: Cannot enter the pores and flow through faster. Small molecules: Enter the pores and move more slowly. Applications: Estimation of protein molecular weight. Desalting protein mixtures. 7  1/20/2025 Ion exchange chromatography Separates proteins based on charge using a column with charged resin. Process: Resin carries either negative (polyanionic) or positive (polycationic) groups. Oppositely charged proteins bind to the resin at the appropriate pH. Proteins are eluted by washing with a salt solution, which disrupts electrostatic interactions. Salt Gradient: Gradually increasing salt concentration releases weakly bound proteins first, followed by tightly bound proteins. Purification techniques Method Principle Separation based on Application Salting-In Stabilizes proteins by trapping Solubility Enhances protein solubility in water molecules around solutions. charged groups at low salt concentrations. Salting-Out High salt concentration reduces Solubility Isolates proteins from complex water availability, causing mixtures. proteins to precipitate. Dialysis Separates proteins from small Molecular Size Removes salts or impurities molecules using a semi- from protein solutions. permeable membrane. Gel Filtration Separates proteins by size; Molecular Size Rough molecular weight larger molecules elute faster estimation and desalting through porous beads. Ion-Exchange Separates proteins by charge Molecular Charge Isolates specific proteins based Chromatography using a column with charged on their charge at a given pH. resin. SDS-PAGE Denatures proteins and Molecular Size Determines purity and separates them by size in an molecular weight of proteins. electric field. 8