Moleculeoftheday

3. Membrane-bounded organelles? Usually structures confined to eukaryotic cells. Made up of a lumen (space) filled with fluid. Materials of lumen kept separate from cytoplasm by membranes.

4. Cellular organelles

5. Inclusions: non-membrane-bounded structures (ribosomes & centrioles)

6. Plasma membrane: {phospholipid bilayer, integral and peripheral proteins, cholesterol}

7. Vesicles: single membrane surrounding a lumen {e.g., lysosomes and microbodies. . . 0.5-1.0 µm}

8. Endoplasmic reticulum (rough ER & smooth ER)

9. Golgi Apparatus: stacked cisternae

10. Mitochondria: two membrane organelle

14. Strategy for isolating cellular organelles 1. Select appropriate source of cells. 2. Place intact cells in a protected environment. 3. Break open cells. 4. Separate organelle of interest from other components.

15. 1. Select an appropriate source of cells.For chloroplasts, choose green leaves. Choose a plant from which it is easy to extract intact organelles. Discard, where feasible, plant parts that don’t contain chloroplasts.

16. 2. Place cells in a protected environment A) Osmotic support: surround cell with solution that provides an osmolarity as large or larger than the osmolarity of the lumen [0.40 M sucrose or 0.25 M NaCl]. Avoid disrupting organelle membranes by avoiding hypotonic solutions

17. Review of Osmosis terms movement of water across a selectively permeable membrane ISOTONIC: SAME SOLUTE CONCENTRATION HYPERTONIC: HIGH SOLUTE CONCENTRATION HYPOTONIC: LOW SOLUTE CONCENTRATION

19. B) pH and ionic composition: Maintain buffered pH around 7.4...divalent cations at a concentration of 10 mM stabilize membranes. Mg++ stabilizes enzyme activities of some membrane proteins.

20. More support... C) Reducing agents: keep oxygen from oxidizing sensitive molecules. D) Chelators: Bind metal ions that may be cofactors for hydrolyzing enzymes. [e.g., EDTA binds copper ions]* *Beer, canned beans, Mcdonald’s special sauce, blue colored shampoos

21. *EDTA a common preservative found in beer, canned beans, Mcdonald’s special sauce, blue colored shampoos

22. Additional support... E) Temperature: protocols call for temperatures between 4o and 0o C. Rationale: i. Enzymes less active at low temperatures. ii. Enzymes of interest remain more stable. iii. Microorganisms that feed on organic molecules slowed down by cold temperature.

23. 3. Break Open Cells Need to disrupt cell but not damage organelles. A) No cell wall-use hypotonic solution. B) Cells with cell wall can be ground open with mortar & pestle or blender. C) Use cell homogenizer to shear plasma membranes. D) Pressure chamber or Sonic oscillation E) Chemical treatments [selective hydrolyzing enzymes]

24. 4. Separate Organelle of Interest Centrifugation: Separate components by density. Organelles larger than 0.5 µm in diameter are often separated by centrifugation. Sample tubes attached to a rotor are spun at high revolutions creating high g forces (e.g., 1000 X g). Denser components settle faster than the less dense. Know what’s in your pellet and supernatant!

25. Sample Calculation rpm = 20,000 r = 7 cm Relative Centrifugal Force (RCF) RCF = (1.119 x 10-5)(rpm)2(r) = (1.119 x 10-5)(20,000)2(7 cm) = 32,000 x g

26. Differential centrifugation Cell extract first subjected to slow speed for a short time to pellet out heavy cellular debris. Pellet thrown out and supernatant kept.

27. Differential centrifugation Supernatant subjected to higher speed and longer time to pellet out organelle of interest. Pellet kept supernatant thrown out.

28. Calculate Chlorophyll Concentrations [Chl] = [Chla] + [Chlb] mg Chl/mL = mg Chla/mL + mg Chlb/mL mg Chl/mL = {(A645) x (0.020)} + {(A663) x (0.0080)} mg Chla/mL = {(A663) x (0.013)} + {(A645) x (0.0027)} mg Chlb/mL = {(A645) x (0.023)} + {(A663) x (0.0047)}

29. Measurement of absorption spectrum from 400 nm to 720 nm Use scanning spectrophotometer to determine the absorption spectrum of the chlorophyll extract. Blue and red light are colors most useful as energy for light reaction in chlorophyll.

30. Visible Light

31. Chlorophyll a Absorbance

32. Background... Only chlorophyll a participates directly in the light reactions of photosynthesis. Other pigments absorb energy and transfer it to chlorophyll a Pigments clustered in the thylakoid membranes

33. Chlorophyll:major light-trapping pigment in most green cells Chlorophyll a (grass-green) the major photosynthetic pigment Accessory pigments Chlorophyll b (yellow-green) Carotenoids Carotenes (red to yellow) Xanthophylls (yellow)

34. Accessory pigments, Phycobilins, occur in red and blue-green algae Phycoerythrin: major pigment in red algae Phycocyanin: major pigment in blue-green algae Pigments conjugated to specific water soluble proteins

35. Chromatography Broad range of procedures used to separate a mixture of molecules or ions.

36. Characteristics of Chromatography MOBILE PHASE: Moving fluid (gas or liquid) Flows by the… IMMOBILE PHASE: (liquid or solid)

37. ORGANIC/AQUEOUS EXTRACTION Separate chloroplast extract with organic solvents and silica by differences in polarity using TLC. Separate proteins by differences in electronegativity using DEAE-cellulose.

38. PREPARATION of Lipids FOR TLC Chromatography Mixture of lipids dissolved in small volume of volatile organic solvent.

39. PREPARATION of Lipids FOR TLC Chromatography Solution placed on a streak parallel to edge of flat sheet of adsorbent material. Volatile solvent evaporates. Lipid mixture remains adsorbed to sheet.

40. Composition of two phases chosen so… Every molecule in the mixture has some attraction for each phase. Each molecule spends some fraction of time in the mobile phase. Each molecule spends some fraction of time in the immobile phase. Relative attraction between the 2 phases is different for each molecule in the mixture.

41. As the mixture of molecules in one phase flow past the other phase… Each molecule in mixture may remain in original phase. Each molecule in mixture may move to the other phase. Time molecule spends in a phase depends on which phase most strongly attracts it.

42. Molecules separated by different relative attraction for one of the phases… Molecules with the stronger relative attraction for the mobile phase will move fastest. Molecules with the stronger relative attraction for the immobile phase will move slowest.

43. Chromatography sheet material Paper chromatography adsorbent sheet is a piece of paper. Thin layer chromatography (TLC) adsorbent sheet is a thin layer of fine silica powder (pure sand) spread uniformly over an inert support such as a sheet of glass or plastic.

44. Separation of adsorbed lipids by TLC 1. Closed chamber prepared with liquid solvent in bottom. 2. Flat sheet with adsorbed lipids is placed in chamber.

45. Separation of adsorbed lipids by TLC 3. Adsorbent material in sheet attracts solvent and solvent creeps up material. 4. Lipids of varying solubility swept along with the solvent.

46. Developed Chromatogram Chromatogram removed from chamber when fastest moving lipid nears top end of the sheet. Separated lipids removed from adsorbent sheet and dissolved in liquid.

47. Sample Chromatogram {Assume adsorbent more polar than solvent} 1. Which band moved the fastest? 2. Which band has the relatively most polar molecules? 3. Which band had greater affinity for the mobile phase?

48. Sample Chromatogram {Assume adsorbent more polar than solvent} 1. Which band moved the fastest? A 2. Which band has the relatively most polar molecules? C 3. Which band had greater affinity for the mobile phase?A

49. Column Chromatography Immobile phase (DEAE) placed in a column. Immobile phase restricted to the column but liquid phase can run through the column and out the bottom. Concentrated mixture of molecules is dissolved in a small volume of solvent (salt/buffer). Solution placed in top of column and liquid percolates down through the immobile phase. Anions stick to DEAE Anions of interest washed out with high concentrations of salt-buffer solutions.

50. Ion exchange resins Made up of two parts Three-dimensional matrix Chemically bonded charged groups within and on the surface of the matrix Resins can be made from many things: Polystyrene, acrylic resins, polysaccharides, and cellulose

51. Quick Review Anion (negative ion, Cl-) Cation (positive ion, Na+)

52. Ion-exchanger classification Cation exchanger: negatively charged functional groups and exchanges positive ions. (e.g., SP-Sephadex) Anionic exchanger: positively charged functional groups and exchanges negative ions. (e.g., DEAE-cellulose)

53. DEAE-cellulose Functional group: diethylaminoethyl Matrix: cellulose Class: weak anionic exchanger

54. Ion Exchange Chromatography Column packed with immobile phase (DEAE cellulose) Surrounded with salt/buffer ions

55. Loading the column Top of column loaded with solution. Mixture of ions and molecules.

56. Separation of Proteins

57. Negatively charged proteins bind to DEAE-cellulose Solute ions with charge opposite of DEAE bind to ion-exchange media. Other ions and molecules flow through column.

58. Proteins bound to DEAE Strength of binding depends upon size of charge and density of charge of solute.

59. Releasing bound proteins Elute column with buffer of increased ionic strength. Different salt concentrations used to release proteins.

60. Collection of elution fractions… Proteins of interest are replaced by ions in salt solution. Proteins extracted from bottom of column.

61. Which protein sample was the more electronegative? Those eluted in the first fractions? Those eluted in the second fractions?

62. fini

63. Supplemental information

64. “Absorption differs from adsorption in that the absorbed substance permeates the bulk of the absorbing substance.” Absorbtion: The taking up, especially by capillary , osmotic, solvent, or chemical action. Adsorbtion: The taking up of one substance at the surface of another.

65. Osmolarity Solute concentration expressed as molarity For non-dissociating molecules, osmolarity roughly equal to molar concentration e.g., 0.11 M sucrose solution = osmolarity of 0.10 Osmolarity is approximately equal to the sum of the osmolarity of each kind of particle in solution Osmolarity within each organelle about equal to that of cytoplasmic matrix

66. Organic molecules that can be separated: Lipids Amino acids Proteins Sugars (mono and disaccharides) Polysaccharides Nucleotides Nucleic acids

67. Factors Determining Movement of Lipids in Mobile Phase 1. Most soluble lipids move fastest. 2. If solvent relatively nonpolar and adsorbent material is more polar, then least polar lipids migrate faster and more polar lipids migrate slower. 3. Faster moving lipids separate from the slower moving lipids, spacing out the molecules from each other along the sheet.

68. Class of Ion-Exchange Resins Classified according to ionizing strength of functional group. Strong Weak

69. Ion Exchange Column Chromatography Immobile phase: DEAE-cellulose Immobile phase carries electrical charges and attracts molecules with net opposite charges. DEAE-cellulose has fixed (+) charges. Negatively charged molecules attracted to DEAE-cellulose (the anionic exchanger). Positive or uncharged molecules flow through the column.

70. Dislodging Protein Molecules After negatively charged proteins have adhered to the DEAE-cellulose, salt/buffer solution of different concentrations are used to displace the proteins. Weakly adhering protein molecules can be dislodged with dilute salt solutions that have (-)ions with stronger negative charges. Stronger adhering proteins can be dislodged with more concentrated salt solutions.