Students analyze spinach pigments and chloroplasts using paper chromatography, a colorimeter, and a spectrometer to understand how plants capture light for photosynthesis.
#1 Whatman Chromatography paper - 10 to 12 cm
0.1 M phosphate buffer - 4 mL
Chloroplast suspension - 2 mL
Chromatography solvent - 5 mL
Cuvettes (with colorimeter) - 5
Distilled water - 13 mL
DPIP in small amber bottle - 3 mL
Floodlight, 100 watt
Glass jar, 10-12 cm tall
Graduated disposable pipet 1-mL - 2
Heat sink (large beaker or flask filled with water)
Ice and water - 1 L
Roll of aluminum foil - 1 per class
Spinach - 1 leaf
The major antenna pigments in algae include chlorophylls, phycobiliproteins and carotenoids and the variation in the composition and relative abundance of these pigments give algae their distinctive colour.
Carbonic anhydrase is an enzyme that interconverts carbon dioxide and hydrogencarbonate which supply Rubisco with carbon dioxide from the pool of HCO3−.
Algae grow faster and are very efficient in absorbing and converting solar energy into chemical energy which is mainly in the form of triacylglycerols.
Students extract pigments from spinach leaves for analysis using chromatography and colorimetry. Paper chromatography separates the pigments present in the extract so they can be identified. Analysis of the extract with a colorimeter allows students to determine the relative absorbance of four different colors of light (blue, green, orange, and red). They relate the chromatography results to the colorimeter measurements to refine their understanding of how plants capture light for photosynthesis. If available, a spectrometer allows students to view the full absorbance spectrum for spinach leaves.
D. von WETTSTEIN and R. P. OLIVER (Carlsberg Laboratorium, Copenhagen,1985) summarized all results and were thus able to develop a modelthat explains the topology of the single protein complexes.
This that one proton is transferred through themembrane the ATP synthase complex. Instead is a newly formedproton given off into solution at one side while another protonis captured and neutralized (by a OH- ion) at the other side ofthe membrane.
In 1961 proposed P. MITCHELL (Glynn Research Laboratories, GreatBritain) that the energy set free during the electron transportis conserved as a across the membrane. The energywould then not be stored as a chemical bond but as an electrochemicalgradient. The electrochemical potential of this gradient wouldbe harnessed to synthesize ATP. The hypothesis explains severalkey observations:
How does this work? It might have been assumed that the electrontransport chain serves the production of energy-rich intermediatesand that these constitute an energy store for the production ofATP. Two arguments against this idea exist:
Since quite some time has the ATP synthase been ascribed the functionof a coupling factor. This means that it is able to utilize thefree energy released by electron transport. Such energy conservationis referred to as or .
A second, independent attempt was and is the use of specific probeslike fluorescence-tagged antibodies that help to find out whethera certain protein (or part of a polypeptide chain) is locatedat the inside or the outside of a membrane. The use of antibodiesagainst specific proteins allows, too, to precipitate these proteinsselectively since only they are able to form the extremely specificantigen - antibody complex.
The research into the proteins essential for photosynthesis startedvery late. The reason is that all of them are membrane-bound whichrendered it nearly impossible to isolate and characterize themwith the classical methods of protein analysis.
The requirements for energy transformation are even higher: completelyintact membranes that are impermeable for protons and that enclosecompartments thus maintaining a stable electrochemical gradientbetween inside and outside. The production of ATP is based ona directed proton dislocation paralleled by a change of the compartment'spH and of its membrane potential.
Students learn how to separate plant pigments using paper chromatography and how to measure the rate of photosynthesis in chloroplast suspensions.