Globular Proteins

1. Protein can be classified according to solubility, shape or the presence of nonprotein groups, etc. • For example: • Solubility, two major families are the globular and fibrous protein. The globular proteins are compact, are roughly spherical or ovoid in shape, and have axial ratios of not over 3 (the ratio of their shortes to longest dimention). • Composition. For example: glycoproteins, lipoproteins, metaloproteins (that incorporate a metal ion such as many enzyme do) etc. • Biologycal functions: enzymes, hormones, neurotransmitters, toxin, contractile muscle (myosin and actin), storage protein (casein, ovalbumin and ferritin), transfort protein (hemoglobin), structural proteins (collagen, elastine, and protein cell membranes) and protective proteins. Amino Acid and Protein

2. Globular Proteins • Myoglobin, a monomeric protein of red muscle, stores oxygen. • Hemoglobin, a tetramic (22) protein of eritrocytes, transport O2 to the tissue and return CO2 and rptons to the lung. Despite different primary structures, the secondary-tertiary structure of subunits of hemoglobin (Hb S), Val replaces the 6 Glu of Hb A. The genetic defect has known as thalassemia result from theh partial or total absence of one or more or  chains of hemoglobin. Amino Acid and Protein

3. Amino Acids and the Primary Stucture of Proteins Important biological functions of proteins 1. Enzymes, the biochemical catalysts 2. Storage and transport of biochemical molecules 3. Physical cell support and shape (tubulin, actin, collagen) 4. Mechanical movement (flagella, mitosis, muscles) (continued) Chapter 3

4. Globular proteins • Usually water soluble, compact, roughly spherical • Hydrophobic interior, hydrophilic surface • Globular proteins include enzymes,carrier and regulatory proteins Chapter 3

5. Fibrous proteins • Provide mechanical support • Often assembled into large cables or threads • a-Keratins: major components of hair and nails • Collagen: major component of tendons, skin, bones and teeth Chapter 3

6. MYOGLOBIN STRUCTURE Amino Acid and Protein

7. Amino Acid and Protein

8. Fibrous Proteins • Collagen is the most abundant of the fibrous proteins that constitute more than 25% of the protein mass in the human body. These proteins in bone, teeth, tendons, skin, and soft connective tissue. Collagen forms a unique triple helix. Every third amino acid residue in collagen is a glycine residue. Collagen is also rich in proline and hydroxyproline, yielding a repetitive Gly-X-Y pattern in which Y generally is proline or hydroxyproline (Gly-X-Y-Gly-X-Y-Gly-X-Y-). Disease of collagen maturation include the vitamin C deficiency disease scurvy and Ehlers-Danlos syndrome. Amino Acid and Protein

9. Amino Acid and Protein

10. Prions-Protein • Human prionrelated protein, PrP, a glycoprotein encoded on the short arm of chromosome 20, normally is monomeric and rich  helix. Pathologic prion proteins, known as PrPc, is rich in  sheet with many hydrophobic aminoacyl side chains. Prion disease are protein conformation diseases transmitted by altering the conformation, fatal neurogenerative diseases characterized by spongiform changes. • For example: Creutzfeld-Jacob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy (mad cow disease) in cattle. Amino Acid and Protein

11. ELECTROPHORESIS Analysis of Biomolecules Amino Acid and Protein

12. H O R – C – C O– NH2 H O R – C – C OH NH3+ H O R – C – C O– NH3+ OH– OH– Anionic form charge -1 pH IEP Zwitter ion form charge 0 pH IEP Cationic form charge +1 pH IEP H+ H+ B. Amphoteric properties • Amino acids are amphoteric molecules ; that is, they have both basic and acidic groups • Monoamino-monocarboxylic acids exist in solution neutral pH are predominantly dipolar ions (or zwitter ion). In dipolar form of an amino acid, the amino group is protonated and positively charged (-NH3+) and the carboxyl group is dissociated and negatively charged (-COO-) Amino Acid and Protein continued

13. H R C NH2 C = O OH Basic Concepts Fundamental to electrophoretical separations is the fact that proteins are electrically charges particles. The charges are derived from amino acids with ionogenic side groups. Amino group Acid group Amino Acid and Protein

14. ELECTROPHORESIS • Electrophoresis is a method for analysis (this is separation or isolation) and characterization of biological polymers. • Here is the use of electrophoresis for the separation of proteins. • The sample containing the proteins to be separated is placed in an electric fieldwhichforces the electrically charged proteins to move. Amino Acid and Protein

15. ELECTROPHORESIS • The movement of molecules (in an electric field) influenced by the Size Charge Shape Chemical composition of the molecule Amino Acid and Protein

16. SEPARATION OF PROTEINS • The separation is normally performed not in free solution but in a supporting gel medium. • The gel can either act as an ”inert” support for the electrophoresis buffer or actively participate in the separation by interacting with the proteins • In the latter case the protein-gel interaction is the actual separation factor while the electrical field merely makes the proteins migrate through the gel Amino Acid and Protein

17. Methods of Electrophoresis • The major difference between methods is the type of support medium, which can be either cellulose or thin gels • 1. Cellulose is used as a support medium for low-molecular weight biochemicals such as amino acids and carbohydrates • 2. Polyacrylamide and agarose gels are widely used as support media for larger molecules. Amino Acid and Protein

18. Methods of electrophoresis divided into 1. Polyacrylamide Gel Electrophoresis (PAGE) 2. Discontinuous Gel Electrophoresis 3. Sodium Dodecyl Sulfate-PAGE (SDS-PAGE) 4. Pulsed Field Gel Electrophoresis (PFGE) 5. Isoelectric Focusing of Protein (IEF) 6. Capillary Electrophoresis (CE) 7. Immunoelectrophoresis (IE) Amino Acid and Protein