This method can help us to understand key questions in photosynthesis research, such as the fade of captured light energy and light harvesting complexes, its kinetics, and efficiency of transfer. The main advantage of this technique is that liposomes provide a membrane compartment resembling the native state of a thylakoid membrane, allowing for studies of ion and pH changes during photosynthesis. To begin the protocol, centrifuge previously-prepared C.meneghiniana cells at 4, 300 times G in a pre-cooled rotor at four degrees Celsius with 500 milliliter centrifuge vials for 15 minutes.
Resuspend the cell pellets homogenization buffer by shaking and pipetting. Transfer the suspension to a single suitable plastic tube and restrict the final volume to 12 milliliters. Next, pre-cool the bead mill and equipment for about 60 minutes.
Fill the 50 milliliter beaker up to 75%with a glass bead mixture, and add the cell suspension. Stir it with a spatula or glass rod and add HB if necessary. For cell disruption, use seven by 45 second pulses at full speed, with 30 seconds of cooling between each pulse.
Filter the disrupted cells over a glass filter funnel and wash them by pouring the HB over the glass beads until they appear clear. Then, pool the wash fraction with the filtrate and keep the final volume lower than 150 milliliters. Centrifuge the sample for 15 minutes at 140 times G using three 50-milliliter plastic tubes to pellet the cell debris.
Carefully transfer the supernatant to 20 milliliter polycarbonate ultracentrifugation vials and discard the pellet. Fill the vials with HB.Equilibrate the weight. Centrifuge in a suitable rotor for one hour at 300, 000 times G and four degrees Celsius to pellet the thylakoid membranes.
Resuspend the membrane pellet with as little washing buffer as possible, using a small painter's brush. Fill the polycarbonate ultracentrifugation vials with washing buffer. Equilibrate their weight.
Centrifuge them for 20 minutes at 200, 000 times G and four degrees Celsius. Resuspend the washed membranes with the painter's brush. Pool all the thylakoids in one 15 milliliter sample vial.
Fill ultracentrifugation tubes with sucrose gradient solution to the top for a volume of one milliliter. Freeze the tubes at minus 20 degrees Celsius until they are completely frozen. Then, allow the tubes to thaw at four degrees Celsius.
Next, use the samples corresponding to 125 micrograms of chlorophyll and adjust them with buffer B1 to a volume of 0.9 milliliters. For solubilization, add N.Dodecyl Beta D maltopyranoside to a final concentration of 20 millimolar. Invert the tube three times.
Place it on ice for 20 minutes with gentle shaking to prevent foam formation. Centrifuge for five minutes at 12, 000 times G in a pre-cooled tabletop centrifuge at four degrees Celsius. Load the supernatant on the gradient, and do not load more than 125 micrograms of total chlorophyll per gradient if 17 milliliter vials are used.
Centrifuge for 22 hours at 100, 000 times G and four degrees Celsius. Recover the desired brown FCP fractions from the gradient using a syringe. Estimate the volume.
Then, remove a five microliter aliquot and dilute it with 995 microliters of B1A. Then, measure the volume of the recovered sample with a five milliliter pipette. Wash the FCP complexes by adding twice the recovered volume with B1 buffer that is free of detergents.
Concentrate the samples in a membrane concentrator with a 30 kilodalton cutoff at 1, 000 times G and four degrees Celsius to an absorbance at 672 nanometers of at least 20. Pipette the liquids into a two milliliter reaction tube and evaporate the chloroform using a gentle nitrogen flow and spread the lipids over the whole area of the tube base. Let the nitrogen flow until all the solvent is evaporated.
Solubilize the lipid mixture in 29 microliters of N Octyl beta d glucopyranoside at four degrees Celsius for four hours. Incubate the lipid mixture for 10 minutes at 30 degrees Celsius. Then, incubate the lipid mixture in a sonicator bath at 25 degrees Celsius for three times for three minutes.
Place the mixture on ice for 30 seconds between each sonication. Add 221 microliters of tricine buffer and 250 microliters of four-fold dialysis buffer. Use an extruder with 0.1 micron polycarbonate membranes to obtain a defined liposome diameter of 50-70 nanometers.
Assemble the extruder with the membrane and filter support. Rinse and moisten the equipment, and work carefully and slowly to prevent air bubbles, and tighten the assembly thoroughly. Fill a syringe with four-fold dialysis buffer and pre-wet the extruder until no bubbles can be seen in the second syringe.
Apply the lipid detergent micelles to the extruder and press the solution from one syringe to the other back and forth. Repeat this step five times, until the solution appears homogenous. Move on to incorporation of the FCP-complexes and add FCP equal to 20 micrograms of chlorophyll a in a total volume of 500 microliters of B1A buffer to 250 microliters of the extruded lipid micelles plus 250 microliters four-fold DP.Incubate the samples for three intervals for three minutes each at 25 degrees Celsius in a thermomixer at 1, 500-3, 000 RPM, interrupted by a 30 second pause on ice.
Cut the lid of four 1.5 milliliter reaction tubes just under the top, giving a ring which still fits on the lid. Prepare 1.5 by 1.5 centimeter pieces of dialysis membranes and wash them in 20 milliliters of one-fold dialysis buffer. Fill each lid with 250 microliters of the sample.
Carefully lay the membrane on the lid so that the compartment is completely filled with the sample and no air bubbles occur. Tighten the reaction tube ring on the assembly so the compartment is closed. Dialyze the samples in 50 milliliters of one-fold dialysis buffer overnight for 12-16 hours on ice on a tumbling shaker.
Replace the buffer with fresh dialysis buffer and add seven milligrams of adsorbent beads to remove the remaining detergents for at least six hours. Replace the dialysis buffer again, and dialyze the samples for another 12 hours. Recover the liposomes by piercing the dialysis membrane with a 200 microliter micropipette tip and aspirate all liposomes from the reaction tube lid.
Centrifuge the FCP liposomes in at least two milliliters of 1X dialysis buffer for 1.5 hours at 100, 000 times G and four degrees Celsius. Recover the liposomes by turning the centrifuge tube at an angle of 45 degrees. Allow the liposomes to move down for one minute.
Recover the FCP liposomes in a final volume of 25-50 microliters, and avoid disturbing the precipitate. Differences in the normalized absorbance spectra indicate pigment loss, which occurs in the spectral region between 500 and 550 nanometers, where carotenoids absorb. Pigment loss is acceptable if the complexes are still functional, indicated by similar emission and excitation spectra.
The chlorophyll fluorescence yield arising from FCP in liposomes is reduced due to excitonic interaction in FCP clusters. The impact of a potassium ion gradient on FCP fluorescence yield was tested in the presence of standard buffer, which drops by 30 percent if potassium chloride is added. The potassium gradient was released by valinomycin, which restores the FCP fluorescence to the initial level and corroborates the hypothesis that FCP complexes respond to potassium ion concentration gradients in vivo.
Following this procedure, other methods like tamrasol fluorescence, pump-probe, and single molecule spectroscopy may be applied in order to study additional questions in light energy transference.
