Light Reactions in Isolated Chloroplasts
University of Minnesota Minneapolis, Minnesota
If isolated chloroplasts are boiled, the enzymes that occur in photosynthesis are disrupted and the DCIP will not reduce.
Chloroplasts are a type of plastid found within the plant they are the basis of photosynthesis. Without photosynthesis there would be no life on earth. Photosynthesis takes the energy from sunlight and coverts it into a chemical energy that can be used by the plant. Isolating chloroplast is a way to separate plastids in order to study the details of the single cell. Isolating the chloroplast helps better understand the functions and helps determine if the isolated chloroplasts are adequate to complete photosynthesis.
Biological activity will be determined by using a dye, dichlorophenolindophenol (DCIP), as a final electron acceptor. Using spectrophotometry, the reduction of DCIP will be measured to follow the dye from an oxidized (DCIP) to a reduced state (DCIPH2) as it
accepts electrons from photosystem I (1). Boiling affects the
Materials and Methods
In order to isolate the chloroplasts, the process had to have been completed rapidly in subdued light. When the solutions were not being used they were stored on ice.
Ten spinach leaves were rinsed and deveined. They were then placed over ice and chopped with a razor. After, the spinach was placed between eight layers of
cheesecloth to squeeze all the liquid derived from the spinach. The liquid was then diluted by adding 20mL of homogenizing buffer (HB). The solution was added to a pre-cooled conical centrifuge tube, and was then centrifuged at 1000 x g for one minute. After, the solution was transferred to another pre-cooled centrifuge tube and was centrifuged at 3000 x g for one minute. Centrifugation resulted in the deposition of a green pellet on the wall of the centrifuge tube near the surface of the liquid (2). This green pellet, together with the...
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