Question

Why would chloroplasts (in solution) placed under the light have a lower absorbance compared to the chloroplasts (in solution) in the dark? What is happening at the molecular level to be showing these results?

The solution will be prepared with: water, DCIP, reaction buffer, and chloroplasts

Please provide a lot of explanation! Thanks!

Answer 1

ANSWER:

• This is an experiment related to the light dependent reactions of photosynthesis, which take place in the chloroplasts.
• DCIP (2,6-dichlorophenolindophenol) a dye which is blue in its oxidized form and colourless in its reduced form is used in the experiment as an artificial electron acceptor.
• In vivo or in the plant the final electron acceptor is NADP⁺, while in the experiment this work is done by DCIP.

• Isolated chloroplasts in the solution when exposed to light would continue to perform Hill reaction. Robert Hill (1937) showed that isolated chloroplasts can evolve oxygen and his finding indicates that the source of the electrons in the light reactions was water. Hence the Hill reaction can be defined as the photoreduction of an electron acceptor by the hydrogen of water, with the evolution of oxygen. This can be written as:

H₂O   +     DCIP (oxidised, blue colour)   ----light-------> DCIP (reduced, colourless)   +    1/2O₂  

In vivo, the Hill reaction is:

H₂O   +    NADP ----light-------> NADPH +    1/2O₂ + H⁺

• As it is well known the Hill Reaction depends on electrons released during the light dependent stage of photosynthesis and picked up by the electron acceptor DCIP (oxidised) and get reduced.
• This reaction can occur only when chloroplast kept in light and oxidised blue dye DCIP gets reduced (colourless) by accepting electrons.
• The rate of the Hill reaction can be measured by taking absorbance reading.

Since the oxidized (blue) DCIP becomes in the reduced state in the tube having chloroplasts under the light, it has lower absorbance reading.

There is no effects in the DCIP state that present in tube having chloroplasts (in solution) placed in the dark.