bio 120
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BIO 120 Postlab Discussions:Ex. 5-8
Jae Rodriguez
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Ex.5: Donnan Equilibrium and Active TransportDonnan Equilibrium
• Two fluid compartments with mixture of charged particles
• Semi-permeable membrane
• One compartment has non-diffusible particles
• Sets electric gradient• Non-uniform distribution of
diffusible particles• Until both sides are
electrically neutral
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Donnan Equilibrium and Active Transport
Active Transport
• Goes against conc. and electric gradients• Requires energy• e.g. Sodium-Potassium Pump• 3 Na+ out, 2 K+ in
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Donnan Equilibrium and Active Transport
Methodology
Donnan Equilibrium(1)pH 3.0 H2O (w/HCl) + 5% gelatin
mixture placed in a dialysis bag(2)Immersed in pH 3.0 H2O(3)pH change in surrounding medium
monitored (5 min intervals for 90 mins)(4)Final pH inside the bag recorded after
90 mins
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Donnan Equilibrium and Active Transport
Active TransportSet-up A(1)Ringer’s soln placed in toad skin bag
(inverted) and weighed(2)Immersed in Ringer’s soln(3)Change in weight monitored (5 min
intervals for 90 mins)
Set-up B(4)Same procedures as above with NaCN in
both bag and medium
(5) Active transport rates computed for both set-ups A and B (based on weight)
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Donnan Equilibrium and Active Transport
Purpose of placing gelatin in the artificial membrane?Simulates negatively charged proteins inside
the cell
Comparison of H+ concentration before and after immersion in acidified H2O
H+ outside go in due to electric and conc. gradientCl- attracted to go in due to conc. gradient but repelled by electric gradient generated by the nondiffusible gelatinoutside the bag [H+] decreases, pH increasesinside the bag [H+] increases, pH decreaseswill continue until equilibrium is reached Donnan Equilibrium
Purpose of Ringer’s soln?Physiological salt for amphibiansNa+ Cl-, K+ Cl-, Ca+ Cl-Keeps the cells alive
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Donnan Equilibrium and Active Transport
Effect of Cyanide on weight of the bagCyanide is inhibitor of Cytochrome C
Oxidase – last enz in ETCNo ATP producedNo energy to drive active transportNo change in the weight of the bag
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Ex.6: Hill Reaction
• Reduction of an electron acceptor by e- from H2O
• Evolution of O2• 2H2O + 2A >> 2AH2 + O2
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Hill Reaction
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Hill Reaction
Dichlorophenolindophenol
• can be used as e- acceptor in place of NADP in vitro• rate of reduction = rate of Hill reaction
Oxidized Reduced
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Hill Reaction
Methodology
(1)Chlorophyll extracted (2 rounds of centrifugation)(2)Read absorbance of chlorophyll at 652 nm(3) Det conc. using Lambert-Beer eq.(4) Dilute to 0.05 mg chl/ml conc(5)Prepare set-ups (table 2, p. 71) – spectro is at 605
nm (6) Monitor absorbance in control and light set-ups
every 5 mins for 30 mins; dark set-up: initial and final only
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Hill Reaction
Expected results
Control – no change in absorbance
Light – progressive decrease in absorbance
Dark – no change in absorbance
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Hill Reaction
Rationaleall soln’s and glasswares precooled:Centrifuge rotors and blender generate
heat. Prevents denaturation of enzymesRates of reactions are maximized
0.35 M NaCl usedhypertonic solncauses plasmolysiscell shriveleasier cell breakage/lysisrelease of subcellular components
Pellet 1 discardedcontains heavier organelles and lipid
fragments
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Hill Reaction
Rationalesuspension centrifuged at 1400 x g for 15
minchloroplasts relatively lightweightwill sediment at this RCF
blank contained chloroplaststo eliminate absorbance of chloroplasts in
the mixture only absorbance of DPIP is of interest
chloroplast susp diluted to 0.05 mg chl/ mlto control levels of DPIP reductiontoo high conc = all DPIP will be consumed
fast
2 different blanksunheated chloroplasts absorb light
differently from heated ones
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Hill Reaction
Why 652 nm in det amt of chl? Why 605 nm in all mixtures?Chlorophyll maximally absorbs light at 652 nm (red)Hence appears green
All mixtures (except blanks) contain DPIPDPIP maximally absorbs light at 605 nm (orange)Hence appears blue
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Hill Reaction
Effect of Light on Hill reactionPhotons drive the transfer of e- in the light dependent reactionsHence necessary to ultimately reduce the electron acceptor
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Ex.7: Cellular Respiration
• Energy (ATP) is extracted from carbohydrates, fats and proteins
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Cellular Respiration
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Ex.7: Cellular Respiration
• Succinate + FAD >> Fumarate + FADH2 <<
• In vitro, DPIP could replace FAD
Methodology
(1) Obtain liver homogenate(2) Prepare set-ups (table 3, p. 79)(3) measure DPIP reduction by
spectrophotometry (use 605 nm)
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Cellular Respiration
What the set-ups represent?Tube 1: w/ substrate and enz, no inhibitor
respiration
Tube 2: w/ substrate and enz, but with inhibitorNaCN inhibits ETCNADH accumulatesinhibits succinate dehydrogenaselittle to no respiration
Tube 3: no substrate, w/ enzymeno respiration
Tube 4: enz is denaturedno respiration
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Cellular Respiration
What the set-ups represent?Tube 5: with competitive inhibitor
slight respiration
Tube 6: with competitive inhibitorincreased conc. of subtratemoderate respiration
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Ex.8: Protein Separation by PAGEProtein Electrophoresis
• Separation of protein molecules
• In an electric field• Separation is based on MW
and electric charge• SDS PAGE• Native Page
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