Core Practical 11 - Hill Reaction Flashcards
What role does NADP play in photosystem I during photosynthesis?
NADP acts as an electron acceptor, accepting electrons and a proton released from split water molecules, causing it to become reduced. Oxygen is released in the process.
This reaction is called the Hill reaction, named after British biologist Robert Hill, who discovered it.
How can the rate of the Hill reaction be investigated?
By adding a redox indicator dye, such as DCPIP, to isolated chloroplasts. The dye acts as an electron acceptor and gets reduced during the light-dependent reaction, causing a color change from blue to colorless.
What equipment is needed for the experiment to measure the Hill reaction?
A colorimeter, which measures how much light a solution absorbs when a light source is shone through it.
What is the first step in preparing the chloroplast extract?
Cut a few leaves (spinach works well) into pieces, removing any tough stalks, and grind them with chilled isolation solution.
What is the purpose of the sucrose in the isolation solution?
Sucrose is included to prevent water from being drawn from the chloroplasts by osmosis.
What should be done after centrifuging the mixture?
Remove the liquid from the top of the tubes, leaving the chloroplast pellets at the bottom.
What is done with the chloroplast pellets after centrifugation?
Re-suspend the pellets in fresh, chilled isolation solution and store it on ice for the rest of the experiment.
How is the colorimeter prepared for the experiment?
Set up the colorimeter with a red filter and zero it using a cuvette containing the chloroplast extract and distilled water.
What should be done with the test tubes during the experiment?
Place a test tube in a rack at a set distance from a bench lamp, add a set volume of chloroplast extract and DCPIP, and mix the contents.
How often should absorbance be recorded during the experiment?
Record the absorbance every 2 minutes for the next 10 minutes.
What indicates the rate of the Hill reaction in the experiment?
The absorbance will decrease as DCPIP gets reduced and loses its blue color; a faster decrease indicates a faster rate of the Hill reaction.
What should be included as negative controls in the experiment?
One control should contain only DCPIP and chilled isolation solution, and the second should contain both DCPIP and chloroplast extract wrapped in tin foil to block light.
What do the negative controls demonstrate?
They show that the DCPIP color change only occurs because of the action of the chloroplasts and that the color of DCPIP doesn’t naturally deteriorate over time.
