EX' 6: Chloroplast Isolation and Separation of Biological Molecules by Chromatography

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ethylaminoethyl-cellulose-more-fun-with-starch/
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EX. 6 Chloroplast Isolation and Separation of
Biological Molecules by Chromatography

• Extraction of membrane-bounded organelles from
  cells.
• Extraction of lipid pigments from chloroplasts.
• Organic separation of non-polar lipids with Thin
  Layer Chromatography.
• Aqueous separation of colored water-soluble
  proteins contained in cell extracts of
  photosynthetic prokaryotes by DEAE anion exchange
  column chromatography.

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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.
  

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Cellular organelles
Plant cell
Animal cell
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Inclusions non-membrane-bounded structures
(ribosomes centrioles)
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Plasma membrane phospholipid bilayer, integral
and peripheral proteins, cholesterol
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Vesicles single membrane surrounding a lumen
e.g., lysosomes and microbodies. . . 0.5-1.0 µm
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Endoplasmic reticulum (rough ER smooth ER)
Ribosomes (inclusions)
ER membrane
Cisternal space
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Golgi Apparatus stacked cisternae
lumen
vesicle
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Mitochondria two membrane organelle
Outer membrane
Inner membrane
Intermembrane space
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Chloroplast plastid family of related plant
organelles
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Cellular plasma membrane
Cell wall
Inner outer membranes
1 µm
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Granum stack of thylakoids
stroma
Thylakoid
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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.

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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 dont
  contain chloroplasts.

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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

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Review of Osmosis terms

• movement of water across a selectively permeable
  membrane
• ISOTONIC SAME SOLUTE CONCENTRATION
• HYPERTONIC HIGH SOLUTE CONCENTRATION
• HYPOTONIC LOW SOLUTE CONCENTRATION

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Isotonic solute equal inside and outside cell,
no net flow of water out or in cell/organelle
Hypertonic solute outside membrane greater
than inside, net outflow of water, cell/organelle
becomes flaccid
Hypotonic solute outside membrane less than
inside, net inflow of water, cell/organelle
bursts!
Osmosismovement of water across a selectively
permeable membrane
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• 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.

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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, Mcdonalds special sauce,
  blue colored shampoos

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EDTA a common preservative found in beer, canned
beans, Mcdonalds special sauce, blue colored
shampoos
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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.

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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

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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 whats in your pellet and supernatant!

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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

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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.

supernatant
pellet
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Differential centrifugation

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

supernatant
pellet
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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)

McKinney, G. (1941). Absorption of light by
chlorophyll solutions. Journal of Biochemical
Chemistry, 140, 315-322.
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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.

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Visible Light
Campbell, N.A. (1990). Biology.
Benjamine/Cummings p. 210
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Chlorophyll a Absorbance
Campbell, N.A. (1990). Biology.
Benjamine/Cummings p. 211
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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

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Chlorophyllmajor 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)

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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

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Chromatography

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

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Characteristics of Chromatography

• MOBILE PHASE
  Moving fluid (gas or liquid)
• Flows by the
• IMMOBILE PHASE
  (liquid or solid)
  

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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.

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PREPARATION of Lipids FOR TLC Chromatography

• Mixture of lipids dissolved in small volume of
  volatile organic solvent.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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?

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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

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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.

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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

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Quick Review

• Anion (negative ion, Cl-)
• Cation (positive ion, Na)

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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)

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DEAE-cellulose

• Functional group diethylaminoethyl
• Matrix cellulose
• Class weak anionic exchanger

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Ion Exchange Chromatography

• Column packed with immobile phase (DEAE
  cellulose)
• Surrounded with salt/buffer ions

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Loading the column

• Top of column loaded with solution.
• Mixture of ions and molecules.

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Separation of Proteins

• Water soluble proteins carry their own unique
  distribution of charges.
• Proteins carrying a net positive charge not
  attracted to DEAE-cellulose, so not separated.
• Proteins carrying a net negative charge can be
  separated with DEAE-cellulose.

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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.

-
-
-
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Proteins bound to DEAE

• Strength of binding depends upon size of charge
  and density of charge of solute.

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Releasing bound proteins

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

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Collection of elution fractions

• Proteins of interest are replaced by ions in salt
  solution.
• Proteins extracted from bottom of column.

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Which protein sample was the more electronegative?

• Those eluted in the first fractions?
• Those eluted in the second fractions?

A. Early fraction
B. Later fraction
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fini
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Supplemental information
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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.

(1996). Concise Science Dictionary, Oxford
University Press, p.4
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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

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Organic molecules that can be separated

• Lipids
• Amino acids
• Proteins
• Sugars (mono and disaccharides)
• Polysaccharides
• Nucleotides
• Nucleic acids

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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.

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Class of Ion-Exchange Resins

• Classified according to ionizing strength of
  functional group.
• Strong
• Weak

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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.

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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.