BIO 120 Postlab Discussions: Ex.

5-8

Jae Rodriguez

Ex.5: Donnan Equilibrium and Active Transport

Donnan Equilibrium Two fluid compartments with mixture of charged particles Semi-permeable membrane One compartment has nondiffusible particles Sets electric gradient Non-uniform distribution of diffusible particles Until both sides are electrically neutral

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

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

Donnan Equilibrium and Active Transport

Active Transport Set-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)

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. gradient Cl- attracted to go in due to conc. gradient but repelled by electric gradient generated by the nondiffusible gelatin outside the bag [H+] decreases, pH increases inside the bag [H+] increases, pH decreases will continue until equilibrium is reached Donnan Equilibrium Purpose of Ringer s soln? Physiological salt for amphibians Na+ Cl-, K+ Cl-, Ca+ ClKeeps the cells alive

Donnan Equilibrium and Active Transport

Effect of Cyanide on weight of the bag Cyanide is inhibitor of Cytochrome C Oxidase last enz in ETC No ATP produced No energy to drive active transport No change in the weight of the bag

Ex.6: Hill Reaction

Reduction of an electron acceptor by e- from H2O Evolution of O2 2H2O + 2A >> 2AH2 + O2

Hill Reaction

Hill Reaction
Dichlorophenolindophenol can be used as e- acceptor in place of NADP in vitro rate of reduction = rate of Hill reaction

Oxidized

Reduced

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

Hill Reaction
Expected results Control no change in absorbance Light progressive decrease in absorbance Dark no change in absorbance

Hill Reaction
Rationale all soln s and glasswares precooled: Centrifuge rotors and blender generate heat. Prevents denaturation of enzymes Rates of reactions are maximized 0.35 M NaCl used hypertonic soln causes plasmolysis cell shrivel easier cell breakage/lysis release of subcellular components Pellet 1 discarded contains heavier organelles and lipid fragments

Hill Reaction
Rationale suspension centrifuged at 1400 x g for 15 min chloroplasts relatively lightweight will sediment at this RCF blank contained chloroplasts to eliminate absorbance of chloroplasts in the mixture only absorbance of DPIP is of interest chloroplast susp diluted to 0.05 mg chl/ ml to control levels of DPIP reduction too high conc = all DPIP will be consumed fast 2 different blanks unheated chloroplasts absorb light differently from heated ones

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 DPIP DPIP maximally absorbs light at 605 nm (orange) Hence appears blue

Hill Reaction
Effect of Light on Hill reaction Photons drive the transfer of e- in the light dependent reactions Hence necessary to ultimately reduce the electron acceptor

Ex.7: Cellular Respiration

Energy (ATP) is extracted from carbohydrates, fats and proteins

Cellular Respiration

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)

Cellular Respiration
What the set-ups represent? Tube 1: w/ substrate and enz, no inhibitor respiration Tube 2: w/ substrate and enz, but with inhibitor NaCN inhibits ETC NADH accumulates inhibits succinate dehydrogenase little to no respiration Tube 3: no substrate, w/ enzyme no respiration Tube 4: enz is denatured no respiration

Cellular Respiration
What the set-ups represent? Tube 5: with competitive inhibitor slight respiration

Tube 6: with competitive inhibitor increased conc. of subtrate moderate respiration

Ex.8: Protein Separation by PAGE

Protein 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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