BIO 120 Postlab Discussions:Ex. 5-8

Jae Rodriguez

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

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 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)

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

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

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

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

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

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

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

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 inhibitorNaCN inhibits ETCNADH accumulatesinhibits succinate dehydrogenaselittle to no respiration

Tube 3: no substrate, w/ enzymeno respiration

Tube 4: enz is denaturedno respiration

Cellular Respiration

What the set-ups represent?Tube 5: with competitive inhibitor

slight respiration

Tube 6: with competitive inhibitorincreased conc. of subtratemoderate respiration

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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