This is an experiment where the effects of light quantity and the effects of DCM

Question

This is an experiment where the effects of light quantity and the effects of DCMU and ammonia on the hill reaction was evaluated using thylakoids 1) DCIP is rather hydrophilic. Why do you think it is necessary to perform this experiment with thylakoids (broken chloroplasts) and not with carefully isolated intact chloroplasts? 2) In any assay, only one or two of the components are actually being detected (sometimes called the detector). What serves as the detector (what changes in a measurable way during the reaction)? How can you be sure the reaction doesn't start before you are ready to read each tube?

Explanation / Answer

The Hill reaction is the light-driven transfer of electrons from water to Hill reagents (non-physiological oxidants) against a chemical potential gradient. The Hill reaction demonstrated that oxygen O2 is produced in plants in a process that is separate from the process that converts carbon dioxide (CO2) to sugars. In the experiment it is demonstrated that isolated chloroplasts would make oxygen (O2) but not fix carbon dioxide (CO2). Hill reagent are dyes that act as artificial electron acceptors during the reaction and changing color when they are reduced. The dye reagent used in this experiment is DCIP. It is blue when oxidized and colorless when reduced.

1) The experiment should be performed with broken chloroplast because the breaking open the organelles' outer membranes and releasing the stroma while leaving the thylakoid membranes intact. If intact chloroplast would be used in the experiment, there will be no reaction, as thylakoid would be not exposed which are the site of the light-dependent reactions of photosynthesis.

2) The Hill reaction occurs at different rates depending on the light intensity supplied to the thylakoids. To measure the reaction rates by taking absorbance readings at different time period. The drop in absorbance over time is taken as a measure of the rate of the Hill reaction.  Thus detector will be the reduction of DCIP with time (which should be linear until DCIP becomes limiting which is indicated by a leveling off of the absorption near the end of the reaction). The maximum rate of DCIP reduction will be the detector point.

3) To be sure about the reaction start point, the experimental tube (Thylakoied +DCIP) colour intensity and absorbance value can be campared to the DCIP control test tube (no thylakoid). They both should be same to ensure that the reaction is not started.

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